Lopinavir and ritonavir for the treatment of cervix disorders

ABSTRACT

The present invention concerns pharmaceutical compositions formulated for topical application that are useful in the treatment of HPV-related pathologies and in particular the treatment of premalignant and malignant conditions of the cervix. The compositions comprise a therapeutically effective amount of lopinavir and ritonavir in a pharmaceutically acceptable vehicle and wherein the weight ratio (w/w) of lopinavir: ritonavir is between 9:1 and 18:1.

Provided herein are methods and compositions for treating and/orinhibiting the development or progression of cancers and benignproliferative disorders of the cervix and particularly such cancers anddisorders caused by human tumour viruses such as human papilloma virus(HPV). In particular compositions are provided comprising lopinavir andritonavir for use in treating and/or inhibiting the progression of HPVrelated dysplasia of the cervix.

BACKGROUND

Many different forms of cancer exist, and it is believed that there aremany different causes of the disease. The incidence of cancer varies,but it represents the second highest cause of mortality, after heartdisease, in most developed countries. Current estimates suggest that onein three Americans alive at present will suffer from some form ofcancer. There is a well-recognised need to develop new and improvedtherapies for treating cancers. Furthermore, there is also a requirementto develop therapeutic agents that may be used to inhibit thedevelopment of cancer in the general population, susceptible high-riskindividuals or as an agent to prevent re-occurrence of disease inindividuals already affected.

Human tumour viruses are recognised to be a major cause of human cancer,and there is a great deal of evidence which supports the contention thatthese viruses cause cancer by inducing genetic instability in infectedcells. Indeed, both the human T-cell leukaemia virus type 1 (HTLV1) Taxand the human papilloma virus type 16 (HPV16) E6 oncoproteins are knownto induce genetic instability producing abnormal numbers of centrosomes,multinucleation and nuclear atypia.

Invasive cervical cancer (ICC) is an example of a cancer associated withviral infection which causes >270,000 deaths per annum with over 85% ofthese occurring in low resource countries. Infection with high-risktypes of HPV has been established as the main aetiological agent forICC. The development of ICC can take 10-20 years and is preceded by HPVrelated pre-invasive pathology which is characterised as eitherlow-grade (CIN1) or high-grade cervical intraepithelial neoplasia(CIN2/3). Lesions can be screened for by cervical cytology testing wherethey are diagnosed (or graded) as either borderline atypical squamouscells of undetermined significance (ASCUS), low-grade squamousintraepithelial lesions (LSIL) or high-grade squamous intraepitheliallesions (HSIL).

The reduction in ICC related mortality in the developed world has beenlargely dependent on organised cytology screening and similar trends incervical cancer mortality have been achieved by organised single screenand treatment in the rest of the world. However, in the poorer nationslack of resources and health education means that most pre-invasivecervical disease remains undiagnosed and untreated. Thus, whereresources are limited, low-cost screening and treatment options areclearly a high priority.

Current treatment options in clinical practice are either by ablative(destructive) or excisional modalities. Systematic reviews havedemonstrated that these treatment modalities have similar success ratesbut have different morbidities. In the developed world, Large LoopExcision of the Transformation Zone LLETZ (aka loop electrosurgicalexcision procedure—LEEP) is used in most colposcopy clinics. Over 80% ofthese procedures are performed under local analgesia and the whole ofthe transformation zone is available for subsequent histologicalexamination. The procedure is associated with a risk ofprimary/secondary haemorrhage, prolonged discharge, infection and a riskof preterm delivery in subsequent pregnancies. The former side effectscan be problematic particularly in low resource countries. Ablativetreatment in the form of cold coagulation and cryotherapy are oftenadvocated for use in low resource countries since these are low cost,require minimal infrastructure and can be carried out by trainednon-medical health professionals. However, some studies have suggestedthat cryotherapy has a higher failure rate compared to other treatmentmodalities.

There are a variety of locally-applied, non-surgical approaches whichhave been evaluated for the treatment of cervical dysplasia including;photodynamic therapy (PDT); off-license use of the anti-cytomegalovirus(CMV) drug cidofovir; local application of the immune activatorImiquimod and direct application of the cytotoxic drug 5 flurouracil(5FU). Although some of these alternative treatment modalities showpromise, their treatment outcomes are inferior to the reported 80-95%success rates obtained in quality assured colposcopy units.

An effective, inexpensive, non-surgical, self-applied treatment for HPVrelated cervical dysplasia would have great potential particularly inlow resource settings. Furthermore, improved compliance with topicaltreatment would be enhanced, if the side effects are minimised.

A recent advance in the treatment of cancers caused by viruses isdisclosed in WO2015/059485 which describes the protease inhibitors,lopinavir and ritonavir (which had previously been used as orallyingested medicaments for the clinical management of retroviralinfections such as HIV) as being clinically useful for topicaladministration to tissues to prevent or treat malignancies caused byHPV. The authors were particularly surprised to find that soft capsulesof KALETRA® (which were marketed by Abbott/Abbvie for the treatment ofHIV infections by oral administration) can be administered topically(i.e. inserted into the vagina for treatment of the cervix) for theprevention or treatment of cancerous conditions, for the prevention ortreatment of oncogenic viral infections and for the prevention ortreatment of benign proliferative orders.

KALETRA® (or its equivalent LOPIMUNE) is available for oral consumptionas a solution comprising 80 mg lopinavir and 20 mg ritonavir permillilitre or as a soft capsule for oral administration that comprises133.3 mg lopinavir and 33.3 mg ritonavir. In both cases the activepharmaceutical ingredients (APIs) are present in a ratio of 4:1(lopinavir:ritonavir). Given that the authors of WO2015/059485 foundsoft capsules of KALETRA® to be efficacious, they reasoned that a ratioof 4:1 would be optimal for topical use.

The present invention is based on work carried out by the inventors toformulate a bespoke formulation of lopinavir and ritonavir for topicalapplication. They have unexpectedly established that the ratio of APIsfound in known pharmaceutical products comprising lopinavir andritonavir (e.g. LOPIMUNE or KALETRA®) may have efficacy, but are notoptimal for treating and/or inhibiting the development or progression ofcancers and benign proliferative disorders of the cervix and the presentinvention provides compositions comprising optimal ratios oflopinavir:ritonavir for use in treating and/or inhibiting thedevelopment of such conditions.

SUMMARY

Disclosed herein are compositions comprising lopinavir in combinationwith ritonavir for use as a medicament in the treatment of cervicalcancer or benign proliferative disorders of the cervix or in theprevention of the development of such cancers and disorders.

According to a first aspect of the invention there is provided apharmaceutical composition that is formulated for topical applicationcomprising a therapeutically effective amount of lopinavir and ritonavirin a pharmaceutically acceptable vehicle wherein the weight (w/w) ratioof lopinavir: ritonavir is between 9:1 and 18:1.

According to a second aspect of the invention there is provided apharmaceutical composition according to the first aspect of theinvention for use as a medicament in treating and/or inhibiting thedevelopment or progression of cervical cancers and benign proliferativedisorders of the cervix.

According to a third aspect of the invention there is provide a methodof treating and/or inhibiting the development or progression of cervicalcancers and benign proliferative disorders of the cervix in a subject inneed of such treatment or inhibition comprising administering atherapeutically effective amount of a pharmaceutical compositionaccording to the first aspect of the invention to said subject.

It is preferred that the cancer or benign proliferative disorder iscaused by a viral infection, more preferably by an oncogenic virus and,in particular, human tumour viruses such as HPV.

In a preferred embodiment the invention concerns treating a subjecthaving an HPV related dysplasia of the cervix comprising administeringto said subject a therapeutically effective dose of the disclosedpharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurposes of illustrating the disclosed compositions and methods, thereare shown in the drawings exemplary embodiments of the compositions andmethods; however, the compositions and methods are not limited to thespecific embodiments disclosed. In the drawings:

FIG. 1 represents a bar chart illustrating the effects of various weightratios of lopinavir:ritonavir on inducing apoptosis in E6/E7immortalised non-transformed endocervical cells as discussed in Example1;

FIG. 2 represents (A) a bar chart illustrating the effects of variousweight ratios of lopinavir:ritonavir on inducing apoptosis in HPV18 +veHeLA cells as discussed in Example 2; and (B) bar chart illustrating theeffects of a 14:1 w/w ratios of lopinavir:ritonavir on inducingapoptosis in a further experiment conducted with HPV18 +ve HeLA (also asdiscussed in Example 2)

FIG. 3 represents: (A) a bar chart illustrating the effects of 12:1weight ratios of lopinavir:ritonavir on inducing apoptosis in HPV16 +veSiHa cells as discussed in Example 3; and (B) illustrative cytometerresults from which the data in the bar charts are based; and

FIG. 4, is a bar chart illustrating the effects of 10:1 and 12:1 weightratios of lopinavir:ritonavir on inducing apoptosis in HPV16 +veSNU17cells as discussed in Example 4;

FIG. 5 represents: (A) a bar chart illustrating the effects of smallerchanges in the w/w ratios of lopinavir:ritonavir between 11 and 13.5:1on their ability to induce apoptosis in Hela cells and (B) illustrativecytometer results from which the data in the bar charts are based.

FIG. 6 is a bar chart illustrating the effects of smaller changes in thew/w ratios of lopinavir:ritonavir between 11 and 13.5:1 on their abilityto induce apoptosis in SiHa cells

DETAILED DESCRIPTION

The disclosed compositions and methods may be understood more readily byreference to the following detailed description taken in connection withthe accompanying figures, which form a part of this disclosure. It is tobe understood that the disclosed compositions and methods are notlimited to the specific compositions and methods described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed compositions and methods.

Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise. When arange of values is expressed, another embodiment includes from the oneparticular value and/or to the other particular value. Further,reference to values stated in ranges include each and every value withinthat range. All ranges are inclusive and combinable.

It is to be appreciated that certain features of the disclosedcompositions and methods which are, for clarity, described herein in thecontext of separate embodiments, may also be provided in combination ina single embodiment. Conversely, various features of the disclosedcompositions and methods that are, for brevity, described in the contextof a single embodiment, may also be provided separately or in anysubcombination.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

The following abbreviations are used herein: human papilloma virus(HPV); Atypical squamous cells of undetermined significance (ASC-US);Low grade squamous intraepithelial lesion (LSIL); Atypical squamouscells—cannot exclude HSIL (ASC-H); High grade squamous intraepitheliallesion (HSIL); Squamous cell carcinoma (SCC); Abnormal glandular cells(AGC); Cervical intraepithelial neoplasia 1 (CIN1); CervicalIntraepithelial neoplasia 2 (CIN2); Cervical intraepithelial neoplasia 3(CIN3); Carcinoma in situ (CIS); Invasive Cervical Carcinoma (ICC).

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. Furthermore, the term “about” when used in reference tonumerical ranges, cut-offs, or specific values is used to indicate thatthe recited values may vary by up to as much as 10% from the listedvalue. As many of the numerical values used herein are experimentallydetermined, it should be understood by those skilled in the art thatsuch determinations can, and often will, vary among differentexperiments. The values used herein should not be considered undulylimiting by virtue of this inherent variation. Thus, the term “about” isused to encompass variations of ±10% or less, variations of ±5% or less,variations of ±1% or less, variations of ±0.5% or less, or variations of±0.1% or less from the specified value.

As used herein, “treating” and like terms refer to reducing the severityand/or frequency of symptoms, eliminating symptoms and/or the underlyingcause of said symptoms, reducing the frequency or likelihood of symptomsand/or their underlying cause, delaying, preventing and/or slowing theprogression cancers or benign proliferative disorders, and improving orremediating damage caused, directly or indirectly, by the cancers ordisorders.

As used herein, the phrase “therapeutically effective dose” refers to anamount of a composition comprising lopinavir and ritonavir, as describedherein, effective to achieve a particular biological or therapeuticresult such as, but not limited to, biological or therapeutic resultsdisclosed, described, or exemplified herein. The therapeuticallyeffective dose may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of thecomposition to cause a desired response in a subject. Such resultsinclude, but are not limited to, the reduction, remission, and/orregression of the benign or malignant disease or prevention of thedevelopment of the benign or malignant disease, as determined by anymeans suitable in the art.

As used herein, “subject” includes a vertebrate, mammal, domestic animalor preferably a human being.

The “pharmaceutically acceptable vehicle” may be any physiologicalvehicle known to those of ordinary skill in the art useful informulating pharmaceutical compositions. The vehicle is most suitablyone suited for delivery of the Active Pharmaceutical Ingredients (APIs)to a target tissue by topical application.

As used herein, “ovule” refers to a cream or gel containing solid orsemi-solid suppository configured for insertion into the vagina.

Disclosed herein are compositions comprising lopinavir and ritonavir foruse as a medicament in the treatment of cervical cancer or benignproliferative disorders of the cervix (e.g. warts) or in the preventionof the development of cervical cancer.

Lopinavir (CAS# 192725-17-0) is a protease inhibitor chemicallydesignated as [1S-[1R*(R*), 3R*,4R*]]-N-[4-[(2,6-dimethylphenoxy0acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H)-pyrimidineacetamide.It has the molecular formula C₃₇H₄₈N₄O₅ and a molecular weight of628.80.

Ritonavir (CAS# 155214-67-5) is a protease inhibitor chemicallydesignated as10-Hydroxy-2-methyl-5-(1-methylethl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid,5-thiazolylmethylester, [5S-(5R*,8R*,10R8,11R*)]. It has the molecularformula C₃₇H₄₈N₆O₅S₂ and a molecular weight of 720.95.

Surprisingly, in view of the prior art, the inventors have found thatmolar ratios of lopinavir:ritonavir for treating, or preventing thedevelopment, of cervical cancer or benign proliferative disorders of thecervix or for prevention of the development of such cancers or suchdisorders should optimally be in the molar ratio range of 10:1-18:1. Therange may be 10.5:1-18:1. Preferably the range is 11.5:1-17:1, morepreferably 11.5:1-16.0:1 and even more preferably 11.5:1-15:1 The rangemay be a molar ratio of about 11.75: 1; about 12:1; about 12.5:1; about13:1; about 13.5:1; about 13.75:1; about 14:0:1; or about 14:5:1. In apreferred embodiment the molar ratio of lopinavir:ritonavir is about12:1. In another preferred embodiment the molar ratio oflopinavir:ritonavir is about 13.8:1.

In a preferred embodiment the molar ratio of Lopinavir: ritonavir isbetween 11:1 and 16:1, more preferably between 11:1 and 13:5 and mostpreferably between 11:1 and 12.5:1. Most preferred molar ratios are11:1, 11.5:1 and 12:1.

It will be appreciated that lopinavir has a molecular weight of 628.8daltons and ritonavir has a molecular weight of 720.95 daltons.Accordingly molar ratios and w/w ratios will not be the same and afactor of 0.872 should be applied when converting molar ratios to w/w.Accordingly the inventors have found that w/w ratios oflopinavir:ritonavir for treating, or preventing the development, ofcervical cancer or benign proliferative disorders of the cervix or forprevention of the development of such cancers or such disorders may bein the weight ratio range 9:1-18:1 or 9:1-16:1. For instance the rangemay be 9.5:1-16:1 or 10.0:1-16:1. Preferably the range is 10.0:1-15.0:1,more preferably 10.25:1-14.5:1 and most preferably 10.5:1-13.0:1. Therange may be a w/w ratio of about 10.25:1; about 10.5:1; about 10.75:1;about 11:1; about 11.25:1; about 11.5:1; about 11.75:1; about 12.0:1;about 12.25:1; about 12.5:1; about 12.75: about 13.0:1; about 13.25:1;about 13.5:1; about 13.75:1; about 14.0:1 or about 14:25:1. In apreferred embodiment the w/w ratio of lopinavir:ritonavir is about10.5:1. In a most preferred embodiment the w/w ratio oflopinavir:ritonavir is about 12:1.

The compositions according to the first aspect of the invention areuseful in the treatment of cervical cancer and particularly useful forpreventing the development of such cancers. Accordingly, normal subjects(i.e. subjects with no detectable cancer), subjects with pre-malignantcells or particularly cancer prone subjects may be treated by topicaladministration of compositions according to the invention with a view topreventing the development of cervical cancer.

The invention is applicable particularly, but by no means exclusively,to pre-cancerous conditions of the cervix and cervical cancers caused byoncogenic viruses, e.g. high-risk or even low-risk forms of humanpapilloma viruses (HPVs).

According to a preferred embodiment of the invention, the compositionsmay be administered to treat, and particularly prevent, the developmentof cervical cancer. It is most preferred that the inhibitors are used totreat or prevent the development of cervical cancers caused by HPV(particularly high-risk types of HPV such as HPV16 or HPV 18).

In the developed nations that have cervical screening programs, HPVtesting and cervical cytology are currently in a state of flux. Atpresent, the cervical smear (or Pap test) is usually carried out priorto HPV testing with follow on procedures depending on the resultsobtained. However, depending on geographical location the HPV test maybe given first. Table 1 shows a typical recommended management protocolbased on the combined results of HPV and Pap tests.

TABLE 1 Recommended Management of combined HPV and Pap tests HPV testPap test Management Negative Negative Repeat testing in 5 years AnyNegative Repeat testing in 3 years Negative ASC-US Repeat testing in 3years Negative LSIL Repeat testing in 6-12 months Not performed ASC-USRepeat testing in 6-12 months Positive Negative Repeat testing in 6-12months Not performed LSIL Immediate colposcopy Positive LSIL Immediatecolposcopy Any ASC-H Immediate colposcopy Positive ASC-US Immediatecolposcopy Any HSIL Immediate colposcopy Any SCC Immediate colposcopyAny AGC Immediate colposcopy Table 1 Acronyms: Atypical squamous cellsof undetermined significance (ASC-US); Low grade squamousintraepithelial lesion (LSIL); Atypical squamous cells - cannot excludeHSIL (ASC-H); High grade squamous intraepithelial lesion (HSIL);Squamous cell carcinoma (SCC); Abnormal glandular cells (AGC). Takenfrom Schiffman, M., Solomon, D., Clinical practice. Cervical-cancerscreening with human papillomavirus and cytologic cotesting. N Engl JMed. 2013, 369(24): 2324-31

Pathology results obtained from biopsies taken at colposcopy aredescribed as: Cervical intraepithelial neoplasia 1 (CIN1); CervicalIntraepithelial neoplasia 2 (CIN2); Cervical intraepithelial neoplasia 3(CIN3); Carcinoma in situ (CIS); Invasive Cervical Carcinoma (ICC). HPVnegative CIN1 is clinically equivalent to LSIL and is currently arescreen in 6-12 months (watch and wait).

This recommended management protocol represents the current bestclinical practice for testing women aged >25. It can be seen thatimmediate colposcopy is recommended for any women with LSIL cytology orgreater (HSIL, etc.) in the absence of an HPV test. Women with LSIL whoare shown to be HPV negative, are rescreened in 6-12 months.

A preferred window of opportunity for use of compositions according tothe first aspect of the invention is between the time of initialdiagnosis with HPV positive disease (ASC-US, LSIL, ASC-H, HSIL) untilcolposcopy, which usually takes approximately 2 weeks or longer,depending on waiting times. Based on what is observed at colposcopy, thedecision is then made to either treat with surgery at this visit (Seeand Treat) or take biopsies for pathology which necessitates a furthercolposcopy visit approximately one month later. Clearly, if thecolposcopy visits are timed appropriately, it may advantageously be thattreatment according to the invention removes the need for surgery.

Compositions according to the invention are not only useful for treatingactual cancers but are also surprisingly useful for preventing thedevelopment of cancer, particularly in patients that may exhibit any ofthe previously described pre-cancerous lesions in HPV-positive patients.Accordingly, the compositions comprising lopinavir and ritonavir may beadvantageously used as a prophylactic.

The compositions may be given to subjects with a genetic disposition todeveloping cervical carcinomas or even those facing environmental risk(e.g. people exposed to carcinogens). In a preferred embodiment, thecompositions may be given to women who are at risk of developingcervical cancer. Such women can include those who have been diagnosed ashaving a high-risk HPV infection (e.g. HPV16 or HPV 18) of theurino-genital tract (and particularly the cervix). At the time ofdiagnosis, there may not be any clinical evidence that such women have acervical carcinoma or even precancerous cells of the cervix, yet womenwith such infections are believed to be at risk of developing cervicalcancer. The compositions may be topically applied to the cervix of womenwith a viral infection of the cervix with a view to treating the viralinfection and thereby preventing the development of cancer at a futuredate.

The compositions may be used to prevent or treat cancer as a monotherapy(i.e. use of the two inhibitors alone) or in combination with othercompounds or treatments used in cancer therapy (e.g. chemotherapeuticagents, radiotherapy).

It is most preferred that the compositions are used to treat humans(i.e. women with or at risk of developing cervical cancer). However, itwill be appreciated that the compositions may also have some veterinaryuse.

Pharmaceutical Compositions

Compositions according to the invention are formulated as a medicamentthat is suitable for topical application and in particular as amedicament formulated for intravaginal delivery and topical applicationto the cervix.

Suitable formulations include, but are not limited to, a gel, cream,paste, ointment, lotion, ovule, soft capsule, suppository, pessary, orany combination thereof. In some aspects, the pharmaceutical compositioncan be formulated as a gel. In some aspects, the pharmaceuticalcomposition can be formulated as a cream. In some aspects, thepharmaceutical composition can be formulated as a paste. In someaspects, the pharmaceutical composition can be formulated as anointment. In some aspects, the pharmaceutical composition can beformulated as a lotion. In some aspects, the pharmaceutical compositioncan be formulated as an ovule. In some aspects, the pharmaceuticalcomposition can be formulated as a soft capsule. In some aspects, thepharmaceutical composition can be formulated as a suppository. In someaspects, the pharmaceutical composition can be formulated as a pessary.In some aspects, the pharmaceutical composition can be formulated as anycombination of the above formulations.

In preferred embodiments, the composition is formulated such that it issuitable for topical delivery of the APIs to the cervix (e.g. as anointment, gel, paste, cream, soft capsule, or pessary) for preventingthe development of, or treating, cervical cancer (e.g. caused byhigh-risk types of HPV such as HPV16 or HPV18).

When used to treat (or prevent the development of) cervical cancer, thecompositions can be formulated as gels, pastes, creams or ointments thatmay be applied directly to the cervix by techniques known to the art.Alternatively, the compositions may be formulated as a vaginalsuppository (or incorporated within a pessary) according to techniquesknown to the art.

In other aspects, the medicaments may be in the form of an ovule. Theovule can comprise a cream or gel located within a coating, the coatingconfigured to melt and release the cream or gel upon being administeredintravaginally. Alternatively, the ovule can consist of a cream or gelthat is configured to melt upon being administered intravaginally.

In one embodiment, the pharmaceutically acceptable vehicle can be aliquid and the composition can be a solution. In another embodiment, thevehicle can be a gel and the composition can be a suppository orpessary. In a further embodiment, the vehicle can be an emulsion (orother pharmaceutically acceptable base) and the composition can be acream or paste. In a further embodiment, the vehicle can be smooth andoily and the composition can be an ointment.

Preferred compositions for use according to the invention are formulatedfor use as vaginal ointments comprising lopinavir and ritonavir in theweight ratios defined for compositions according to the first aspect ofthe invention.

Liquid vehicles may be used in preparing solutions, suspensions,emulsions, syrups, elixirs and pressurized compositions. The activeingredient can be dissolved or suspended in a pharmaceuticallyacceptable liquid vehicle such as water, an organic solvent, a mixtureof both or pharmaceutically acceptable oils or fats. The liquid vehiclecan contain other suitable pharmaceutical additives such assolubilizers, emulsifiers, buffers, preservatives, suspending agents,thickening agents, colours, viscosity regulators, stabilizers orosmo-regulators. Suitable examples of liquid vehicles include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).The vehicle can also be an oily ester such as ethyl oleate and isopropylmyristate. The liquid vehicle for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellent.

The pharmaceutical composition may be a vaginal suppository.Conventional vehicles, coatings and other constituents of vaginalsuppositories may be chosen by one skilled in the art to form asuppository that is characterised by the fact it comprisestherapeutically effective amounts of lopinavir and ritonavir. In oneembodiment vaginal suppositories typically may use polyethylene glycolas a main vehicle for lopinavir and ritonavir. The balance of thevehicle may be made up of, for example, Oleic acid, PEG 35, castor oil,purified water, gelatin, sorbitol special polyol, or any combinationthereof. Lopinavir and ritonavir are virtually insoluble in water and itis preferred that such organic bases (or equivalents thereof) areformulated in such vaginal suppositories.

Preferred pharmaceutical compositions are ointments and comprisevehicles most suited for preparing ointments. Some of these formats havewater present within the composition. However topical compositions whichcontain water are not always ideal for use with an active pharmaceuticalingredient (API) which is prone to degradation by hydrolysis becausethis may result in a short shelf life of the pharmaceutical productand/or the requirement to store the composition in certain conditions inorder to minimize degradation of the active API. Lopinavir and ritonavirare examples of APIs which can be prone to degradation. Therefore,preferred compositions are anhydrous compositions comprising non-aqueousvehicles. Such vehicles are typically smooth oily compositions andtypically contain a significant proportion (w/w) of pharmaceuticallyacceptable oils or fats (e.g. oleic acid). Such vehicles can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, suspending agents, thickeningagents, colours, viscosity regulators, stabilizers or osmo-regulators.

It will be appreciated that the amount of lopinavir and ritonavir incompositions according to the invention will depend up the exactcomponents and in particular the vehicle for the composition.

By way of example only, suitable amounts of lopinavir in an ointment(e.g. the anhydrous compositions and other preferred compositionsdescribe below) may be from about 0.1-30% w/w. In some embodiments, theamount of lopinavir in the composition can be from about 1.0-25% w/w. Insome embodiments, the amount of lopinavir in the composition can be from2.0-20% w/w. For instance, and ointment may comprise about 5%, 6%, 10%or 12% (w/w) lopinavir.

Suitable amounts of ritonavir in such compositions may be from about0.01-3% w/w. In some embodiments, the amount of ritonavir in thecomposition can be from about 0.1-2.5% w/w. In some embodiments, theamount of ritonavir in the composition can be from 0.15-1.5% w/w. Forinstance, an ointment may comprise about 0.4775%, 0.5%, 0.625%, 0.955%,1% or 1.25% (w/w) ritonavir.

A preferred composition may comprise about 8 to about 14% by weight oflopinavir and about 0.75 to about 1.4% by weight of ritonavir. Forexample, the composition may comprise by weight 10% lopinavir and 0.955%ritonavir or the composition may comprise by weight 12% lopinavir and 1%ritonavir.

Another preferred composition may comprise about 4 to about 7% by weightof lopinavir and about 0.375 to about 0.75% by weight of ritonavir. Forexample, the composition may comprise by weight 5% lopinavir and 0.4775%ritonavir or the composition may comprise by weight 6% weight lopinavirand 0.5% ritonavir.

Preferred Anhydrous Compositions

Preferred compositions for use according to the invention are anhydrouscompositions for topical application comprising:

-   -   a. lopinavir and ritonavir in a weight ratio of between 9:1 and        18:1; and    -   b. a hydrophilic muco-adhesive agent;        wherein upon topical administration of the anhydrous composition        to a site of application the anhydrous composition transforms        into a muco-adhesive composition.

Advantageously, upon topical administration of the anhydrous compositionto a site of application (such as the mucosal membrane of the cervix)the anhydrous composition transforms to a muco-adhesive composition.This in-situ transformation of the anhydrous composition to amuco-adhesive composition results in the muco-adhesive compositionhaving different rheological behaviour, such as an increase inviscosity, and/or an increase in adhesiveness and/or increase intackiness compared to the anhydrous composition.

Preferably the weight ratio of Lopinavir: ritonavir is between 10:5:1and 13.5:1 and most preferably about 12.0:1 (w/w).

The hydrophilic muco-adhesive agent may be a non-ionic polymer or anionic polymer. In one embodiment, the non-ionic polymer is a celluloseether. In one embodiment, the cellulose ether is selected from methylcellulose, ethylcellulose and hydroxypropylmethylcellulose.

In a preferred embodiment, the hydrophilic muco-adhesive agent ishydroxypropylmethylcellulose. In one embodiment, thehydroxypropylmethylcellulose has a degree of methoxy substitution ofbetween 19 and 24% by weight and a degree of hydroxypropyl substitutionof between 4 and 12% by weight.

In another embodiment, the ionic polymer is sodium polyacrylate.

Optionally, additional excipients may be included in the anhydrouscomposition.

In one embodiment, the anhydrous composition further comprises athickener. A thickener is an excipient which when added to a mixtureincreases the viscosity of the mixture and confers the anhydrouscomposition with greater physical stability and/or control duringdelivery of the active pharmaceutical ingredient to the site ofapplication. In one embodiment, the thickener is selected from mono diglyceride, ceresin wax, and hydrogenated vegetable oil, or a combinationthereof.

In one embodiment, the anhydrous composition further comprises astiffening agent. The stiffening agent is an excipient used to stiffenthe composition so that the composition is a semi-solid at roomtemperature. Conveniently, the stiffening agent is a saturated freefatty acid, such as a C₁₀-C₃₈ saturated free fatty acid, such as aC₁₆-C₂₂ saturated free fatty acid. A saturated free fatty acid is a freefatty acid (i.e., the fatty acid is not bound to another molecule, suchas glycerol) wherein there are no double bonds between the carbon atomsin the fatty acid. In one embodiment, the stiffening agent is selectedfrom capric acid, undecylic acid, lauric acid, tridecylic acid, myristicacid, pentadecylic acid, palmitic acid, margaric acid, stearic acid,nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid,tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid,heptacosylic acid, montanic acid, nonacosylic acid, melissic acid,henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid,ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid andoctatriacontanoic acid. The stiffening agent is preferably stearic acid.

In one embodiment, of the total saturated fatty acid (bound and freeform fatty acid) present within the composition, at least 90% by weight,such as at least 95% by weight such as at least 98% by weight, such asat least 99% by weight, or such as at least 99.5% by weight, is in thefree form, i.e., not esterified or bound to other components such asglycerol. The skilled person would be aware of methods used to determinethe free fatty acid content versus the total fatty acid content. Forexample, the free fatty acid content can be measured by reacting thefree fatty acid with a chromogenous compound, thus changing thefrequency that the chromogenous compound absorbs electromagneticradiation. Thus, the concentration of the chromogenous compound reactedcan be determined by monitoring the chromogenous compound using asuitable wavelength which in turn can be used to determine the freefatty acid content in the sample.

In one embodiment, the saturated free fatty acid is not in the form of atriglyceride or polysorbate.

In one embodiment, the anhydrous composition further comprises a solventfor lopinavir and ritonavir.

In one embodiment, the solvent is selected from an unsaturated freefatty acid, PEG castor oil, diethylene glycol monoethyl ether, propyleneglycol, polyethylene glycol, and a medium chain triglyceride. Fattyacids are usually derived from triglycerides or phospholipids. Glycerolhas three hydroxyl functional groups, which can be esterified with one,two, or three fatty acids to form mono-, di-, or triglyceridesrespectively. Phospholipid molecules consist of two hydrophobic fattyacid “tails” and a hydrophilic “head” consisting of a phosphate group.These two components are joined together by a glycerol molecule. Bothtriglyceride and phospholipids comprise fatty acids in a bound state.Conversely, free fatty acids are fatty acids which are not bound, thatis they are not esterified. An unsaturated free fatty acid is a freefatty acid wherein there is at least one double bond between carbonatoms in the fatty acid. In one embodiment, the solvent is anunsaturated free fatty acid.

In one embodiment, the unsaturated free fatty acid is selected fromoleic acid, linoleic acid, alpha-linoleic acid, palmitoleic acid,gondoic acid, and ricinoleic acid.

In one embodiment, the unsaturated free fatty acid is oleic acid.

In one embodiment, of the total unsaturated fatty acid (bound and freeform unsaturated fatty acid) present within the composition, at least90% by weight, such as at least 95% by weight such as at least 98% byweight, such as at least 99% by weight, or such as at least 99.5% byweight, is in the free form, i.e., not esterified or bound to othercomponents such as glycerol. The skilled person would be aware ofmethods used to determine the free fatty acid content versus the totalfatty acid content. For example, the free fatty acid content can bemeasured by reacting the free fatty acid with a chromogenous compound,thus changing the frequency that the chromogenous compound absorbselectromagnetic radiation. Thus, the concentration of the chromogenouscompound reacted can be determined by monitoring the chromogenouscompound using a suitable wavelength which in turn can be used todetermine the free fatty acid content in the sample.

It is to be understood that free fatty acids products that arecommercially available may contain small amounts of other free fattyacids. For example, oleic acid typically contains 7-12% saturated freefatty acids, such as stearic and palmitic acid, together with otherunsaturated free fatty acids, such as linoleic acid (Handbook ofPharmaceutical Excipients, 2^(nd) Edition, see entry for Oleic acid).The terms saturated free fatty acid or unsaturated free fatty acid areto be understood as meaning the saturated free fatty acid or theunsaturated free fatty acid are of Pharmacopeia grade, such as the USPharmacopeia and/or the British Pharmacopeia, and that the saturatedfree fatty acid or unsaturated free fatty acid may contain small amountsof other free fatty acids.

In one embodiment, the unsaturated free fatty acid is not in the form ofa triglyceride or polysorbate.

The total fatty acid (unsaturated and saturated fatty acids in the boundand free form) present within the composition may be at least 90% byweight, such as at least 95% by weight such as at least 98% by weight,such as at least 99% by weight, or such as at least 99.5% by weight, inthe free form, i.e., not esterified or bound to other components such asglycerol.

In one embodiment, the anhydrous composition further comprises oleicacid and stearic acid.

In one embodiment, the anhydrous composition further comprises oleicacid, stearic acid, mono di glyceride, ceresin wax, and hydrogenatedvegetable oil

In one embodiment, there is provided an anhydrous composition fortopical application comprising:

-   -   a. ritonavir;    -   b. lopinavir;    -   c. hydroxypropylmethylcellulose;    -   d. oleic acid;    -   e. stearic acid; and    -   f. butylated hydroxytoluene

In one embodiment, the anhydrous composition comprises:

-   -   a. about 1.2 to about 1.4% by weight of ritonavir;    -   b. about 9 to about 11% by weight of lopinavir;    -   c. about 0.5 to about 1.5% by weight of        hydroxypropylmethylcellulose;    -   d. about 55 to about 65% by weight of oleic acid;    -   e. about 28 to about 32% by weight of stearic acid; and    -   f. about 00.5 to about 0.5% by weight of butylated        hydroxytoluene;        wherein all % are by weight based upon the total weight of the        composition.

In another embodiment, the anhydrous composition comprises:

-   -   a. about 0.5 to about 0.7% by weight of ritonavir;    -   b. about 4 to about 6% by weight of lopinavir;    -   c. about 0.5 to about 1.5% by weight of        hydroxypropylmethylcellulose;    -   d. about 55 to about 65% by weight of oleic acid;    -   e. about 28 to about 32% by weight of stearic acid; and    -   f. about 0.05 to about 0.5% by weight of butylated        hydroxytoluene;        wherein all % are by weight based upon the total weight of the        composition.

A preferred anhydrous composition comprises:

-   -   a. ritonavir;    -   b. lopinavir;    -   c. hydroxypropylmethylcellulose;    -   d. oleic acid;    -   e. stearic acid;    -   f. butylated hydroxytoluene;    -   g. mono diglyceride;    -   h. ceresin wax;    -   i. hydrogenated vegetable oil;

j. polyoxyl 100 stearate; and

k. glycerol monooleate;

In one embodiment, the anhydrous composition comprises:

-   -   a. about 0.9 to about 1.1% by weight of ritonavir;    -   b. about 9 to about 11% by weight of lopinavir;    -   c. about 0.5 to about 1.5% by weight of        hydroxypropylmethylcellulose;    -   d. about 55 to about 65% by weight of oleic acid;    -   e. about 4 to about 5% of stearic acid;    -   f. about 0.1 to about 0.3% by weight of butylated        hydroxytoluene;    -   g. about 4 to about 6% by weight of mono diglyceride;    -   h. about 5 to about 7% by weight of ceresin wax;    -   i. about 9 to about 11% by weight of hydrogenated vegetable oil;    -   j. about 1 to about 3% by weight of polyoxyl 100 stearate; and    -   k. about 2 to about 4% by weight of glycerol monooleate;        wherein all % are by weight based upon the total weight of the        composition

In one embodiment, the anhydrous composition comprises:

-   -   a. about 0.9 to about 1.1% by weight of ritonavir;    -   b. about 11 to about 13% by weight of lopinavir;    -   c. about 0.5 to about 1.5% by weight of        hydroxypropylmethylcellulose;    -   d. about 50 to about 60% by weight of oleic acid;    -   e. about 4 to about 5% of stearic acid;    -   f. about 0.1 to about 0.3% by weight of butylated        hydroxytoluene;    -   g. about 4 to about 6% by weight of mono diglyceride;    -   h. about 5 to about 7% by weight of ceresin wax;    -   i. about 9 to about 11% by weight of hydrogenated vegetable oil;    -   j. about 1 to about 3% by weight of polyoxyl 100 stearate; and    -   k. about 2 to about 4% by weight of glycerol monooleate;        wherein all % are by weight based upon the total weight of the        composition.

In one embodiment, the anhydrous composition comprises:

-   -   a. about 0.4 to about 0.6% by weight of ritonavir;    -   b. about 4 to about 6% by weight of lopinavir;    -   c. about 0.5 to about 1.5% by weight of        hydroxypropylmethylcellulose;    -   d. about 55 to about 65% by weight of oleic acid;    -   e. about 4 to about 5% of stearic acid;    -   f. about 0.1 to about 0.3% by weight of butylated        hydroxytoluene;    -   g. about 4 to about 6% by weight of mono diglyceride;    -   h. about 5 to about 7% by weight of ceresin wax;    -   i. about 9 to about 11% by weight of hydrogenated vegetable oil;    -   j. about 1 to about 3% by weight of polyoxyl 100 stearate; and    -   k. about 2 to about 4% by weight of glycerol monooleate;        wherein all % are by weight based upon the total weight of the        composition.

In one embodiment, the anhydrous composition comprises:

-   -   a. about 0.4 to about 0.6% by weight of ritonavir;    -   b. about 5 to about 7% by weight of lopinavir;    -   c. about 0.5 to about 1.5% by weight of        hydroxypropylmethylcellulose;    -   d. about 55 to about 65% by weight of oleic acid;    -   e. about 4 to about 5% of stearic acid;    -   f. about 0.1 to about 0.3% by weight of butylated        hydroxytoluene;    -   g. about 4 to about 6% by weight of mono diglyceride;    -   h. about 5 to about 7% by weight of ceresin wax;    -   i. about 9 to about 11% by weight of hydrogenated vegetable oil;    -   j. about 1 to about 3% by weight of polyoxyl 100 stearate; and    -   k. about 2 to about 4% by weight of glycerol monooleate;        wherein all % are by weight based upon the total weight of the        composition.

Other Preferred Compositions

Another preferred composition for use according to the invention is apharmaceutical composition comprising:

-   -   a. an unsaturated free fatty acid;    -   b. a stiffening agent; and    -   c. lopinavir and ritonavir in a weight ratio of between 10:1 and        18:1;

wherein the unsaturated free fatty acid is present at a level of atleast 20% by weight of the total pharmaceutical composition weight andwherein the pharmaceutical composition is a semi-solid at roomtemperature.

Preferably the weight ratio of Lopinavir: ritonavir is between 10:5:1and 13.5:1 and most preferably about 12.0:1 (w/w).

Conventional compositions employ vegetable oils and/or polysorbates asagents to thicken the composition. It has been advantageouslyestablished that an unsaturated free fatty acid and a stiffening agentcan be used to prepare a pharmaceutical composition which is asemi-solid at room temperature that can be present in a stationarymaterial state until an external stress is applied resulting in flow ofthe material. Such an external stress can be the application of thecomposition to a target tissue (i.e. the cervix) and the inventors havefound that such compositions are particularly effective for deliveringlopinavir and ritonavir to the target tissue.

Advantageously, the pharmaceutical composition only comprises fats inthe form of free fatty acids (unsaturated free fatty acid and/orsaturated free fatty acid), for example all fatty acids present in thecomposition are in the form of a free fatty acid. This allows thepharmaceutical composition to be manufactured at room temperature whichis advantageous when the at least one active pharmaceutical is prone todegradation, and wherein the rate and/or extent of degradation isincreased when the active pharmaceutical ingredient is exposed to heat.

The unsaturated free fatty acid may be as described above for anhydrouscompositions and is preferably oleic acid.

The stiffening agent may be as described above for anhydrouscompositions and is preferably stearic acid.

Such pharmaceutical compositions may optionally include a muco-adhesiveagent and other excipients as described above for anhydrouscompositions.

Preferred pharmaceutical compositions are disclosed in Example 7 andTables 2-5 and manufactured as disclosed in Example 7 or Example 10.1.

Dosing

It will be appreciated that the amount of lopinavir and ritonavirrequired is determined by biological activity and bioavailability, whichin turn depends, in part, on the precise mode of administration, thephysicochemical properties of the composition employed, and whether thecompositions are being used as a monotherapy or in a combined therapywith other oral or topical medicines. Indeed, it is also possible thatthe at least one active pharmaceutical ingredient could be topicallyapplied in addition to oral dosing of the same compounds or other activepharmaceutical ingredient(s). The frequency of administration will alsobe influenced by the abovementioned factors and particularly thehalf-life of the active pharmaceutical ingredients within the subjectbeing treated.

Daily doses may be given as a single administration (e.g. as anointment, soft capsule, vaginal suppository, ovule or pessary).Preferably the compositions are administered once a day and preferablyin the evening before going to sleep. Alternatively, administration maybe twice or more times during a day. As an example, the compositions maybe topically administered at least once a day, such as once a day ortwice a day.

Optimal dosages to be administered may be determined by those skilled inthe art, and will vary with the strength of the preparation, the mode ofadministration, and the advancement of the disease condition. Additionalfactors depending on the particular subject being treated will result ina need to adjust dosages, including, for example, subject age, weight,diet, and time of administration.

Suitable amounts of lopinavir to be given are a daily dose of betweenabout 0.1 mg to about 10.0 g. In some embodiments, the daily dose oflopinavir can be from about 10 mg to about 5.0 g. In some embodiments,the daily dose of lopinavir can be from about 25 mg to about 1.0 g.Conveniently, the daily dose of lopinavir may be between 25 mg and 500mg (e.g. about 300 mg or 150 mg).

Suitable amounts of ritonavir to be given are a daily dose of about 0.01mg to about 1.0 g. In some embodiments, the daily dose of ritonavir canbe from about 1.0 mg to about 250.0 mg. In some embodiments, the dailydose of ritonavir can be from about 2.5 mg to about 100 mg.Conveniently, the daily dose of ritonavir may be between 5 mg and 50 mg(e.g. about 25 mg, 28.65 mg, 12.5 mg or 14.325 mg).

In one embodiment, about 300 mg of lopinavir and about 28.65 mg ofritonavir per day may be administered to the cervix of a woman.

In another embodiment, about 150 mg of lopinavir and about 14.325 mg ofritonavir per day may be administered to treat the cervix of a woman.

In another embodiment, about 300 mg of lopinavir and about 25 mg ofritonavir per day may be administered to treat the cervix of a woman.

In another embodiment, about 150 mg of lopinavir and about 12.5 mg ofritonavir per day may be administered to treat the cervix of a woman.

It will be appreciated that the amount of a composition which needs tobe administered to a subject will depend upon the concentration oflopinavir and ritonavir in the composition and also the size of thelesion which needs to be treated. By way of example preferred ointments(e.g. preferred anhydrous composition discussed above) may beadministered by a syringe in volumes sufficient to administer 0.5-15.0g, preferably in volumes sufficient to administer 1.0-7.5 g of thecomposition.

In one embodiment about 3.0 g of a composition may be administered to asubject per day. Such dosage forms may comprise about 300 mg oflopinavir and about 28.65 mg of ritonavir; or about 150 mg of lopinavirand about 14.325 mg of ritonavir.

In another embodiment, about 2.5 g of a composition may be administeredto a subject per day. Such dosage forms may comprise about 300 mg oflopinavir and about 25 mg of ritonavir; or about 150 mg of lopinavir andabout 12.5 mg of ritonavir.

In one preferred embodiment about 3.0 g of the ointment disclosed inTable 2 or Table 3 is administered to the cervix, by a syringeapplicator, as a once per day application (preferably in the eveningbefore retiring for the night).

In a most preferred embodiment about 2.5 g of the ointment disclosed inTable 4 or Table 5 is administered to the cervix, by a syringeapplicator, as a once per day application (preferably in the eveningbefore retiring for the night).

Preferred Treatment Regimens

The medicament may be administered to a subject for as long as treatmentis required. The length of time for which treatment will be requiredwill depend upon the exact condition being treated or prevented and itsseverity. A skilled person will appreciate that treatment should bemaintained in view of a number of factors which will include anyrequirement to eradicate any oncogenic virus (e.g. HPV); to reduce oreradicate cells with a precancerous or cancerous phenotype; or to shrinkor eradicate any tumour or other lesion (e.g. a wart).

In one embodiment a course of treatment may be for 2-4 weeks, 7-21 daysor for about 14 days. After this time a clinician may assess whether thecourse of treatment has been successful. A decision may then be madewhether or not to continue treatment.

It will be appreciated that a clinician may wish to take into accountmenstruation when deciding on a treatment regimen for women withconditions relating to the cervix. Accordingly, a preferred treatmentregimen may be for about 14-21 days and can be administered betweenmenses. A clinician may elect to stop topical treatment of the cervixduring menses and recommence a new course of treatment in the nextmenstrual cycle. By way of example, a preferred treatment regimen canbe: (1) 14-21 days of administration; (2) followed by 1-14 days withouttreatment (during which menses may occur if treating the cervix); and(3) a further cycle of 14-21 days of treatment if this is consideredmedically necessary.

In a preferred embodiment, the cervix of a woman may be treated suchthat she receives about 300 mg of lopinavir and about 28.65 mg ritonavirper day for 14-21 days; treatment can then be stopped for 1-14 days anda clinical reassessment can be conducted; then, if necessary a secondtreatment cycle of about 300 mg of lopinavir and about 28.65 mgritonavir per day can be administered for a further 14-21 days. Afterthe second cycle a further clinical assessment can be made and adecision made about whether or not subsequent treatment cycles arerequired.

In another preferred embodiment, the cervix of a woman may be treatedsuch that she receives about 150 mg of lopinavir and about 14.325 mgritonavir per day for 14-21 days; treatment can then be stopped for 1-14days and a clinical reassessment can be conducted; then, if necessary asecond treatment cycle of about 150 mg of lopinavir and about 14.325 mgritonavir per day can be administered for a further 14-21 days. Afterthe second cycle a further clinical assessment can be made and adecision made about whether or not subsequent treatment cycles arerequired.

In another preferred embodiment, the cervix of a woman may be treatedsuch that she receives about 300 mg of lopinavir and about 25 mgritonavir per day for 14-21 days; treatment can then be stopped for 1-14days and a clinical reassessment can be conducted; then, if necessary asecond treatment cycle of about 300 mg of lopinavir and about 25 mgritonavir per day can be administered for a further 14-21 days. Afterthe second cycle a further clinical assessment can be made and adecision made about whether or not subsequent treatment cycles arerequired.

In another preferred embodiment, the cervix of a woman may be treatedsuch that she receives about 150 mg of lopinavir and about 12.5 mgritonavir per day for 14-21 days; treatment can then be stopped for 1-14days and a clinical reassessment can be conducted; then, if necessary asecond treatment cycle of about 150 mg of lopinavir and about 12.5 mgritonavir per day can be administered for a further 14-21 days. Afterthe second cycle a further clinical assessment can be made and adecision made about whether or not subsequent treatment cycles arerequired

Each of these regimens may most preferably involve administering (once aday) to a subject of 0.5-15.0 g of an ointment according to theinvention, preferably 1.0-7.5 g of an ointment and most preferably about2.5 g or about 3.0 g of the ointment.

A preferred treatment regimen is described in Example 8.

Treatment of HPV Related Dysplasia

In preferred embodiments of the invention the pharmaceuticalcompositions may be used to treat female subjects having an HPV relateddysplasia of the cervix.

As used herein, “dysplasia” encompasses pre-invasive lesions and cancer.HPV related pre-invasive lesions include high grade squamousintraepithelial lesion (HSIL), atypical squamous cells of undeterminedsignificance (ASCUS), and low grade squamous intraepithelial lesion(LSIL). HPV related cancers include, for example, cervicalintraepithelial neoplasia (CIN) and invasive cervical cancer (ICC).

The disclosed methods and treatment regimens can be used to treat HPVrelated dysplasia. In some aspects, for example, the disclosed methodsand treatment regimens can be used to treat HSIL. In some aspects, thedisclosed methods and treatment regimens can be used to treat ASCUS. Inother aspects, the disclosed methods and treatment regimens can be usedto treat LSIL. In other aspects, the disclosed methods and treatmentregimens can be used to treat CIN. In yet other embodiments, thedisclosed methods and treatment regimens can be used to treat ICC.Additionally, the disclosed methods and treatment regimens can be usedto inhibit the progression of HPV related dysplasia. In some aspects,for example, the disclosed methods and treatment regimens can be used toinhibit the progression of HSIL. In some aspects, the disclosed methodsand treatment regimens can be used to inhibit the progression of ASCUS.In other aspects, the disclosed methods and treatment regimens can beused to inhibit the progression of LSIL. In other aspects, the disclosedmethods and treatment regimens can be used to inhibit the progression ofCIN. In yet other embodiments, the disclosed methods and treatmentregimens can be used to inhibit the progression of ICC.

The pharmaceutical composition can reduce the severity of the HPVrelated dysplasia. Severity of the HPV related dysplasia can be measuredand graded by, for example, changes in histology. Methods of performinghistology on biopsies of HPV-related lesions are well known in the art.In some embodiments, for example, the disclosed compositions can reducethe severity of CIN 3. In some aspects, the disclosed compositions canreduce the severity of CIN3 to CIN2. In other aspects, the disclosedcompositions can reduce the severity of CIN3 to CIN1. In other aspects,the disclosed compositions can reduce the severity of CIN3 to HPVnegative. In other aspects, the disclosed compositions can reduce theseverity of CIN2 to CIN1. In other aspects, the disclosed compositionscan reduce the severity of CIN2 to HPV negative. In other aspects, thedisclosed compositions can reduce the severity of CIN1 to HPV negative.

|A subject may have a cervical cytology (e.g., from a PAP smear) ofHSIL, ASCUS, or LSIL. Administration of the pharmaceutical compositionto such a subject can reduce the cervical cytology. In some aspects, thecervical cytology is reduced from HSIL to a normal cytology. In someaspects, the cervical cytology is reduced from HSIL to ACSUS. In someaspects, the cervical cytology is reduced from HSIL to LSIL. In someaspects, the cervical cytology is reduced from ACSUS to a normalcytology. In some aspects, the cervical cytology s reduced from LSIL toa normal cytology.

Histological assessments to evaluate and/or grade the severity of theHPV related dysplasia and cytological screening can be performed at anysuitable time period prior to, during, and/or post-treatment with thedisclosed compositions. In some embodiments, the methods furthercomprise post-treatment monitoring of the subject. Suitable time-framesfor post-treatment monitoring include, but are not limited to, 4 weeks,8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36weeks, 40 weeks, 44 weeks, 48 weeks, or 52 weeks following treatmentwith the disclosed pharmaceutical compositions. In some aspects, forexample, a histological assessment can be performed at baseline (priorto treatment) and 6 months post-treatment to assess changes in CINstatus. In other aspects, a cytological screen can be performed atbaseline and 6 months post-treatment to assess changes in cervicalcytology. In yet other aspects, a histological assessment andcytological screen can be performed at baseline and 6 monthspost-treatment to assess changes in CIN status and cervical cytology,respectively.

The extent and grade of an HPV related dysplasia can be reduced during aperiod of from about 4 weeks to about 52 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced from about 4 weeks to about 46 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced from about 4weeks to about 40 weeks following administering said composition. Insome aspects, the extent and histological grade of the dysplasia can bereduced from about 4 weeks to about 34 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced from about 4 weeks to about 28 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced from about 4weeks to about 24 weeks following administering said composition. Insome aspects, the extent and histological grade of the dysplasia can bereduced from about 4 weeks to about 18 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced from about 4 weeks to about 12 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced from about 6weeks to about 10 weeks following administering said composition. Insome aspects, the extent and histological grade of the dysplasia can bereduced from about 8 weeks to about 52 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced from about 12 weeks to about 52 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced from about 18weeks to about 52 weeks following administering said composition. Insome aspects, the extent and histological grade of the dysplasia can bereduced from about 24 weeks to about 52 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced from about 30 weeks to about 52 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced from about 36weeks to about 52 weeks following administering said composition. Insome aspects, the extent and histological grade of the dysplasia can bereduced about 42 weeks to about 52 weeks following administering saidcomposition. In some aspects, the extent and histological grade of thedysplasia can be reduced from about 48 weeks to about 52 weeks followingadministering said composition.

In some aspects, the extent and histological grade of the dysplasia canbe reduced within about 4 weeks following administering compositionsaccording to the invention. In some aspects, the extent and histologicalgrade of the dysplasia can be reduced within about 5 weeks followingadministering said composition. In some aspects, the extent andhistological grade of the dysplasia can be reduced within about 6 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced within about 7weeks following administering said composition. In some aspects, theextent and histological grade of the dysplasia can be reduced withinabout 8 weeks following administering said composition. In some aspects,the extent and histological grade of the dysplasia can be reduced withinabout 9 weeks following administering said composition. In some aspects,the extent and histological grade of the dysplasia can be reduced withinabout 10 weeks following administering said composition. In someaspects, the extent and histological grade of the dysplasia can bereduced the extent and histological grade of the dysplasia can bereduced within about 11 weeks following administering said composition.In some aspects, the extent and histological grade of the dysplasia canbe reduced within about 12 weeks following administering saidcomposition. In some aspects, the extent and histological grade of thedysplasia can be reduced within about 16 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced within about 20 weeks followingadministering said composition. In some aspects, the extent andhistological grade of the dysplasia can be reduced within about 24 weeksfollowing administering said composition. In some aspects, the extentand histological grade of the dysplasia can be reduced within about 28weeks following administering said composition. In some aspects, theextent and histological grade of the dysplasia can be reduced withinabout 32 weeks following administering said composition. In someaspects, the extent and histological grade of the dysplasia can bereduced within about 36 weeks following administering said composition.In some aspects, the extent and histological grade of the dysplasia canbe reduced within about 40 weeks following administering saidcomposition. In some aspects, the extent and histological grade of thedysplasia can be reduced within about 44 weeks following administeringsaid composition. In some aspects, the extent and histological grade ofthe dysplasia can be reduced within about 48 weeks followingadministering said composition. In some aspects, the extent andhistological grade of the dysplasia can be reduced within about 52 weeksfollowing administering said composition.

The cervical cytology grade can similarly be reduced from about 4 weeksto about 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 46 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 40 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 34 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 28 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 24 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 18 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 4 weeks toabout 12 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 6 weeks toabout 10 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 8 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 12 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 18 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 24 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 30 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 36 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 42 weeks toabout 52 weeks following administering said composition. In someaspects, the cervical cytology grade be reduced from about 48 weeks toabout 52 weeks following administering said composition.

In some aspects, the cervical cytology grade reduced within about 4weeks following administering said composition. In some aspects, thecervical cytology grade reduced within about 5 weeks followingadministering said composition. In some aspects, the cervical cytologygrade reduced within about 6 weeks following administering saidcomposition. In some aspects, the cervical cytology grade reduced withinabout 7 weeks following administering said composition. In some aspects,the cervical cytology grade reduced within about 8 weeks followingadministering said composition. In some aspects, the cervical cytologygrade reduced within about 9 weeks following administering saidcomposition. In some aspects, the cervical cytology grade reduced withinabout 10 weeks following administering said composition. In someaspects, the cervical cytology grade reduced within about 11 weeksfollowing administering said composition. In some aspects, the cervicalcytology grade reduced within about 12 weeks following administeringsaid composition. In some aspects, the cervical cytology grade reducedwithin about 16 weeks following administering said composition. In someaspects, the cervical cytology grade reduced within about 20 weeksfollowing administering said composition. In some aspects, the cervicalcytology grade reduced within about 24 weeks following administeringsaid composition. In some aspects, the cervical cytology grade reducedwithin about 28 weeks following administering said composition. In someaspects, the cervical cytology grade reduced within about 32 weeksfollowing administering said composition. In some aspects, the cervicalcytology grade reduced within about 36 weeks following administeringsaid composition. In some aspects, the cervical cytology grade reducedwithin about 40 weeks following administering said composition. In someaspects, the cervical cytology grade reduced within about 44 weeksfollowing administering said composition. In some aspects, the cervicalcytology grade reduced within about 48 weeks following administeringsaid composition. In some aspects, the cervical cytology grade reducedwithin about 52 weeks following administering said composition.

The therapeutically effective dose of the disclosed pharmaceuticalcompositions can be administered for about 1 week to about 4 weeks.After this time a clinician may assess whether the course of treatmenthas been successful. A decision may then be made whether or not tocontinue treatment.

In some embodiments, the composition induces apoptosis of HPV infectedcells.

EXAMPLES Example 1 Assessment of the Effects of 8:1-16:1 W/W Ratios ofLopinavir:Ritonavir at a Total API Concentration of 20 μM on E6/E7Immortalised Non-Transformed Endocervical Cells

E6/E7 endocervical cells are a non-transformed cell line with aphenotype similar to the phenotype found in HPV related cervicaldysplasia. The cell line therefore represents a good model forevaluating the effect of lopinavir and ritonavir on precancerous andearly stage cancerous conditions. For instance, HPV related DysplasiasCIN1-CIN 3.

1.1 Methods 1.1.1 Cell Culture

E6/E7 cells were maintained by standard methods in RPMI growth mediumcontaining 5% Fetal Calf Serum (FCS) at 5% CO₂ and 37° C.Experiments were conducted on cells seeded from a T75 confluent starterculture to a T25 flask. The cells were cultured in the T25 flasks for 6days. The first 3 days followed standard culture conditions whereas fordays 4-6 the cells were grown in growth medium without FCS. This stepsynchronised cells in the growth cycle and the inventors found this stepimproved the performance of subsequent assay steps.

1.1.2 Treatments

After the sixth day (1.1.1), culture was continued for a further 3 daysin RPMI growth medium containing 5% FCS and a total API concentration of20 μM and with lopinavir and ritonavir present in the ratios discussedin the results section.

1.1.3 DNA Fragmentation Assays in a NC3000 Image Cytometer (ChemometecLtd, Norway).

After the final day of culture with the APIs, the cells weretrypsinised, pelleted and washed with PBS using standardized procedures.The cells were then fixed in 70% ethanol for a minimum of 4 hours,washed in PBS, counted and then stained with DAPI according to themanufacturers DNA Fragmentation assay procedure (Chemometec Ltd,Norway).Stained cells were then analyzed using eight chamber slides with theNC3000 Image Cytometer. A representative cell cycle/DNA fragmentationprofile is shown in FIG. 3(B). the cytometer distinguished cells from 4phases of the cell cycle (M1-M4 in FIG. 3(B) which corresponds to Sub G1(dead cells), G1, S and G2) depending on detecting characteristic DNAstaining in each cell in the assayed sample. Each data point on the Boxand Whisker Plots is the product of four such assays which were analyzedfor statistical significance with pairwise permutation test using the Rprogram. The percentage of cells undergoing apoptotic DNA fragmentation(Sub G1) versus those which have intact 2N DNA (G1) was calculated andthe data plotted as bar charts for cells treated with the specifiedratios of lopinavir:ritonavir.

1.2 Results

FIG. 1 represents data presented as the percentage of cells which areundergoing apoptotic DNA fragmentation (Sub G1) versus those which haveintact 2N DNA (G1) following treatment with lopinavir and ritonavir at8:1, 10:1, 12:1, 14:1 and 16:1 (w/w).The inventors were surprised to note that a statistically significantpeak of apoptotic activity (corresponding to induced cell death of theE6/E7 cells) was apparent for a 12:1 (w/w) ratio of the APIs (See FIG.1).This illustrates that lopinavir and ritonavir, in ratios according tothe invention, have improved efficacy for treating pre-invasivepathologies, and particularly HOV-related pathologies, of the cervix(CIN1-CIN3).

Example 2 Assessment of the Effects of 8:1-14:1 W/W Ratios ofLopinavir:Ritonavir at a Total API Concentration of 20 μM on HPV18Positive HeLa Cervical Carcinoma Cells

The effect of different ratios of lopinavir and ritonavir were assessedin HeLa cells. HeLa cells are HPV18 positive cervical carcinoma cellline and are a good model HPV-related malignant disease (e.g. Invasivecervical cancer (ICC)).

2.1 Methods

The methods described in 1.1 were followed except:2.1.1: The cells were cultured in the T25 flasks for 2 days. The firstday followed standard culture conditions whereas for the second thecells were grown in growth medium without FCS.2.1.2: Culture was continued for a further 2 days in RPMI growth mediumcontaining 5% FCS and a total API concentration of 20 μM and withlopinavir and ritonavir present in the ratios discussed in the resultssection.

2.2 Results

FIG. 2(A) represents data presented as the percentage of cells which areundergoing apoptotic DNA fragmentation (Sub G1) versus those which haveintact 2N DNA (G1) following treatment with DMSO (control); 20 μMlopinavir alone; and lopinavir and ritonavir at 8:1, 10:1, 12:1, and14:1 (w/w).The inventors noted that the apoptotic activity caused by lopinavirincreased when ritonavir was included at a ratio of 8:1 and 10:1 and, aswas the case for E6/E7 cells, peaked for a 12:1 (w/w) ratio of the APIs.FIG. 2(B) represents data from a second experiment examining the effectsof DMSO (control); 20 μM lopinavir alone; and lopinavir and ritonavir at8:1 and 14:1 (w/w). The graph illustrates that lopinavir:ritonavir in aratio according to the invention (14:1) was significantly more effectivefor killing HeLa cells than lopinavir alone or the APIs at a ratio of8:1.This illustrates that lopinavir and ritonavir, in ratios according tothe invention, will have improved efficacy for treating HPV-relatedmalignant disease and ICC.

Example 3 Assessment of the Effects of 12:1 W/W Ratios ofLopinavir:Ritonavir at a Total API Concentration of 20 μM on HPV16Positive SiHa Cervical Carcinoma Cells

The effect of different ratios of lopinavir and ritonavir were assessedin SiHa cells. SiHa cells are a HPV16 positive cervical carcinoma cellline and are a good model for HPV-related malignant disease and ICCInvasive cervical cancer (ICC).

3.1 Methods

The methods described in 1.1 were followed except:3.1.1: The cells were cultured in the T25 flasks for 7 days. The 4 daysfollowed standard culture conditions whereas for days 5-7 the cells weregrown in growth medium without FCS.3.1.2: Culture was continued for a further 3 days in RPMI growth mediumcontaining 5% FCS and a total API concentration of 20 μM and withlopinavir and ritonavir present in the ratios discussed in the resultssection.

3.2 Results

FIG. 3(A) represents data presented as the percentage of cells which areundergoing apoptotic DNA fragmentation (Sub G1) versus those which haveintact 2N DNA (G1) following treatment with DMSO (control); 20 μMlopinavir alone; and lopinavir and ritonavir at 8:1 or 12:1 (w/w). Thedata illustrates that lopinavir:ritonavir in a ratio according to theinvention (12:1) was significantly more effective for killing SiHa cellsthan lopinavir alone or the APIs at a ratio of 8:1 (w/w).This illustrates that lopinavir and ritonavir, in ratios according tothe invention, will have improved efficacy for treating HPV-relatedmalignant disease and ICC.FIG. 3(B) represents the cell cycle/DNA fragmentation profile (as Boxand Whisker Plots) generated from the Image Cytometer and is included toillustrate the raw data from which FIG. 3(A) is derived. Similarprofiles (not presented) were the basis for FIGS. 1, 2 and 4.

Example 4 Assessment of the Effects of 10:1 and 12:1 W/W Ratios ofLopinavir:Ritonavir at a Total API Concentration of 25 μM on HPV16Positive SNU17 Cervical Carcinoma Cells

SNU17 cells are HPV16 positive cervical cell carcinoma cell derived froma 40-year-old Mongolian women and were obtained from Creative Bioarray.The cell line is a further model for treating HPV-related malignantdisease and Invasive cervical cancer (ICC).

4.1 Methods

The methods described in 1.1 were followed except:4.1.1: The cells were cultured in the T25 flasks for 4 days. The firsttwo days followed standard culture conditions whereas for days 3 and 4the cells were grown in growth medium without FCS.4.1.2: Culture was continued for a further 3 days in RPMI growth mediumcontaining 5% FCS and a total API concentration of 2504 and withlopinavir and ritonavir present in the ratios discussed in the resultssection.

4.2 Results

FIG. 4 represents data presented as the percentage of cells which areundergoing apoptotic DNA fragmentation (Sub G1) versus those which haveintact 2N DNA (G1) following treatment with DMSO (control); 20 μMlopinavir alone; and lopinavir and ritonavir at 8:1, 10:1 or 12:1 (w/w).The data illustrates that lopinavir:ritonavir in ratios according to theinvention (10:1 and 12:1 (w/w)) are significantly more effective forkilling SNU17 cells than lopinavir alone or the APIs at a ratio of 8:1(w/w).

Example 5 Assessment of the Effects of Small Changes in W/W Ratios(11-13.5) of Lop/Rit at a Total API Concentration of 20 μM on Hela Cells

Additional experiments were conducted to further evaluate the optimalratio of lopinavir:ritonavir for use according to the invention.

5.1 Methods

The methods described in Example 2 were followed.

5.2 Results

FIG. 5(A) represents data presented as the percentage of cells which areundergoing apoptotic DNA fragmentation (Sub G1) versus those which haveintact 2N DNA (G1) following treatment with DMSO (control); 20 μMlopinavir alone; and lopinavir and ritonavir at 11:1, 11:5, 12:1,12.5:1; 13:1 and 13:5 (w/w). The data illustrates thatlopinavir:ritonavir in ratios according to the invention, andparticularly at 11.5 and 12:1, were significantly more effective forkilling HeLa cells than lopinavir alone.This illustrates that lopinavir and ritonavir, in ratios according tothe invention, will have improved efficacy for treating HPV-relatedmalignant disease.FIG. 5(B) represents the cell cycle/DNA fragmentation profile (as Boxand Whisker Plots) generated from the Image Cytometer and is included toillustrate the raw data from which FIG. 5(A) is derived. Similarprofiles (not presented) were the basis for FIGS. 1,2 and 4.

Example 6 Assessment of the Effects of Small Changes in W/W Ratios(11-13.5) of Lop/Rit at a Total API Concentration of 20 μM on SIHA Cells

Further experiments were conducted to further evaluate the optimal ratioof lopinavir:ritonavir for use according to the invention.

6.1 Methods

The methods described in Example 3 were followed.

6.2 Results

FIG. 6 represents data presented as the percentage of cells which areundergoing apoptotic DNA fragmentation (Sub G1) versus those which haveintact 2N DNA (G1) following treatment with DMSO (control); 20 μMlopinavir alone; and lopinavir and ritonavir at 11:1, 11:5, 12:1,12.5:1; 13:1 and 13:5 (w/w). The data illustrates thatlopinavir:ritonavir in a ratios according to the invention, andparticularly at 11:1, 11.5 and 12:1, were significantly more effectivefor killing SiHa cells than lopinavir alone.This illustrates that lopinavir and ritonavir, in ratios according tothe invention, will have improved efficacy for treating HPV-relatedcervical malignant disease or ICC.These data presented in Example 1-6 illustrate that lopinavir andritonavir in ratios according to the invention have significantlygreater effects on induced cell death than lopinavir alone or lopinavirand ritonavir in ratios that fall outside the range defined for thepresent invention. The fact that the preferred API ratios wereefficacious in each of E6/E7 immortalised endocervical, HeLa, SiHa andSNU17 cells clearly demonstrate the usefulness of lopinavir andritonavir, in the defined ratios, for treating HPV-related cervicalpathologies.These data presented in Example 1-6 illustrate that lopinavir andritonavir in ratios according to the invention have significantlygreater effects on induced cell death than lopinavir alone or lopinavirand ritonavir in ratios that fall outside the range defined for thepresent invention. The fact that the preferred API ratios wereefficacious in each of E6/E7 immortalised endocervical, HeLa, SiHa andSNU17 cells clearly demonstrate the usefulness of lopinavir andritonavir, in the defined ratios, for treating cervical pathologies andparticularly HPV-related malignant disease.

Example 7 Preparation of Preferred Formulations

For all formulations present below, all materials used arepharmaceutical grade (either US Pharmacopeia or European Pharmacopeia)except for white ceresin wax, which is Japanese Pharmaceutical Excipientgrade.The manufacture of a vaginal dosage form exhibiting rheology suitablefor syringe applicator vaginal dosing is described below in accordancewith Tables 2-5.

-   -   i. Add into the mixer the following        materials—3,4,5,6,7,8,9,1,10,11    -   ii. Exclude air from the interior of the vessel    -   iii. Heat to 70° C. while low shear mixing, to achieve a clear,        transparent melt.    -   iv. Add into the mixer the following material—2    -   v. Exclude air from the interior of the vessel    -   vi. Mix via low shear, to finely disperse the HPMC within the        melt    -   vii. Reduce the content temperature to 45° C. while low shear        mixing    -   viii. Discharge to storage vessel and exclude air during        storage.    -   ix. Pack composition into aluminium tubes, suitable for        dispensing 1.0-5.0 g of composition.        Aluminium tubes may contain a volume of 20-50 mls of        composition.

TABLE 2 # Ingredient Function(s) % w/w 1 Oleic acid Unsaturated free62.823 fatty acid 2 Hypromellose 2208 Muco-adhesive 1.00 (4000 cps) 3Mono di glycerides (type 1) Thickener 5.00 4 White ceresin wax Thickener6.00 5 Hydrogenated vegetable oil Thickener 10.00 (type 1) 6 Polyoxyl100 stearate Blending agent 2.00 7 Stearic acid Stiffening agent 4.50 8Glycerol monooleater Blending agent 3.00 9 ButylatedhydroxytolueneAntioxidant 0.20 10 Lopinavir API 5.000 11 Ritonavir API 0.4775 TOTAL100.000

TABLE 3 # Ingredient Function(s) % w/w 1 Oleic acid Unsaturated free57.345 fatty acid 2 Hypromellose 2208 Muco-adhesive 1.00 (4000 cps) 3Mono di glycerides (type 1) Thickener 5.00 4 White ceresin wax Thickener6.00 5 Hydrogenated vegetable oil Thickener 10.00 (type 1) 6 Polyoxyl100 stearate Blending agent 2.00 7 Stearic acid Stiffening agent 4.50 8Glycerol monooleater Blending agent 3.00 9 ButylatedhydroxytolueneAntioxidant 0.20 10 Lopinavir API 10.000 11 Ritonavir API 0.9550 TOTAL100.000

TABLE 4 # Ingredient Function(s) % w/w 1 Oleic acid Unsaturated free61.800 fatty acid 2 Hypromellose 2208 Muco-adhesive 1.00 (4000 cps) 3Mono di glycerides (type 1) Thickener 5.00 4 White ceresin wax Thickener6.00 5 Hydrogenated vegetable oil Thickener 10.00 (type 1) 6 Polyoxyl100 stearate Blending agent 2.00 7 Stearic acid Stiffening agent 4.50 8Glycerol monooleater Blending agent 3.00 9 ButylatedhydroxytolueneAntioxidant 0.20 10 Lopinavir API 6.000 11 Ritonavir API 0.500 TOTAL100.000

TABLE 5 # Ingredient Function(s) % w/w 1 Oleic acid Unsaturated free55.300 fatty acid 2 Hypromellose 2208 Muco-adhesive 1.00 (4000 cps) 3Mono di glycerides (type 1) Thickener 5.00 4 White ceresin wax Thickener6.00 5 Hydrogenated vegetable oil Thickener 10.00 (type 1) 6 Polyoxyl100 stearate Blending agent 2.00 7 Stearic acid Stiffening agent 4.50 8Glycerol monooleater Blending agent 3.00 9 ButylatedhydroxytolueneAntioxidant 0.20 10 Lopinavir API 12.000 11 Ritonavir API 1.000 TOTAL100.000The manufacture of a placebo ointment suitable for syringe applicatorvaginal dosing is described below in accordance with Table 6.

-   -   i. Add into the mixer the following materials—3,4,5,6,7,8,9,1,    -   ii. Exclude air from the interior of the vessel    -   iii. Heat to 70° C. while low shear mixing, to achieve a clear,        transparent melt.    -   iv. Add into the mixer the following material—2    -   v. Exclude air from the interior of the vessel    -   vi. Mix via low shear, to finely disperse the HPMC within the        melt    -   vii. Reduce the content temperature to 45° C. while low shear        mixing    -   viii. Discharge to storage vessel and exclude air during        storage.    -   ix. Pack composition into aluminium tubes, suitable for        dispensing 1.0-5.0 g of composition. Aluminium tubes may contain        a volume of 20-50 mls of composition.

TABLE 6 # Ingredient Function(s) % w/w 1 Oleic acid ** Unsaturated free68.30 fatty acid 2 Hypromellose 2208 Muco-adhesive 1.00 (4000 cps) 3Mono di glycerides (type 1) Thickener 5.00 4 White ceresin wax Thickener6.00 5 Hydrogenated vegetable oil Thickener 10.00 (type 1) 6 Polyoxyl100 stearate Blending agent 2.00 7 Stearic acid Stiffening agent 4.50 8Glycerol monooleate* Blending agent 3.00 9 ButylatedhydroxytolueneAntioxidant 0.20 TOTAL 100.00 *Melt completely/mix source supply priorto dispensing ** N2 purge source supply after sampling ***Protect fromUV light

Example 8 A Preferred Dosing Regimen

Subjects were provided with aluminium tubes comprising the compositiondefined in Table 5.Subjects were instructed to apply 2.5 g of the composition to thecervix, once a day and preferably in the evening. The aluminium tubeswere adapted to load disposable syringes with 2.5 g of the composition.After use the syringes were disposed of. The aluminium tube may be usedto load a syringe on subsequent days if the tube still containssufficient composition.Treatment may continue for 14-21 days as recommended by a physician.Treatment may then be stopped (and this halt may be timed to coincidewith menses) for 1-14 days. During this time a clinical reassessment canbe conducted; then, if necessary a further treatment cycle or cycles maybe administered for a further 14-21 days per cycle. After each cycle afurther clinical assessment can be made, and a decision made aboutwhether subsequent treatment cycles are required.

Example 9 A Phase 1, Single Centre, Double Blind, Randomised, Parallelgroup, Ascending Single and Multiple Dose, Safety and Tolerability,Pharmacokinetic (PK) and Pharmacodynamic (PD) Study of PreferredFormulations in Healthy Women Volunteers

The compositions according to Tables 2 and 3 were investigated accordingto the clinical trial described below (and compared to the placebo ofTable 6).

9.1 Methods Study Objectives

-   -   1. To evaluate the safety, PK and PD of compositions in healthy        women volunteers after multiple doses of Formulations according        to Tables 2 and 3.    -   2. To observe the rates of side effects reported by women using        the compositions compared to placebo.

Investigational Plan/Study Design

This study comprises two cohorts, both in healthy volunteers with nocervical pathology. There were 9 participants per Cohort, of whom 6received active and 3 receive placebo. For all formulations tested, theamount of the composition administered per dose is 3 g. For compositionof Table 3 this equates to 300 mg of lopinavir and 28.7 mg of ritonavirbeing administered to the patients per dose. For the composition ofTable 2 this equates to 150 mg of lopinavir and 14.3 mg of ritonaviradministered per dose.

Cohort 1:

Period 1: Single dose of composition of Table 2 or placebo followed byconfinement. PK blood sampling during confinement.Period 2: 21 daily doses of composition of Table 2 or placebo followedby PK blood sampling.

Cohort 2:

Period 1: Single dose of composition of Table 3 or placebo followed byconfinement. PK blood sampling during confinement.Period 2: 21 daily doses of composition of Table 3 or placebo followedby PK blood sampling.

Participation Criteria Inclusion Criteria:

-   -   a. Women, 20 to 45 years old, with an intact uterus and vagina.    -   b. Generally, in good health with no clinically significant        pulmonary, cardiac, gastroenterological, pancreatic, neurologic,        renal, musculoskeletal, rheumatologic, metabolic, neoplastic, or        endocrine disease.    -   c. BMI of =>19 and <=30.0    -   d. ECG and vital signs within normal ranges    -   e. Agree to no Alcohol from 48 hours prior to dosing in period 1        until 7 days after receiving the final dose in period 2.    -   f. Abstain from food or beverages containing grapefruit,        starfruit, pomegranate, pineapple, or pomelo for the entire        study    -   g. Able and willing to abstain from sexual intercourse +/−6        hours around dosing within Periods 1 and 2    -   h. Able and willing to use stringent methods of contraception        after required abstinence period through to Day 29 (7 days after        receiving the final dose in period 2), including the use of a        non-latex condom (for partner protection) and a second        acceptable contraception method; vasectomy, contraceptive pill,        contraceptive implants or IUDs are allowed. (note: IUDs should        have been inserted at least 1 month prior to enrolment and not        because of the involvement in this study)    -   i. Agree to abstain from activities such as vaginal douching or        insertion of any vaginal products other than the study drug for        at least 48 hours prior to enrolment and throughout the study.    -   j. Negative Pap test at screening or within 3 years of enrolment        and no history of cervical intraepithelial lesions within the        previous 3 years    -   k. Able and willing to return to the clinic for all study        procedures.    -   l. Able and willing to provide informed consent.

Exclusion Criteria:

-   -   a. Women who are pregnant, plan to become pregnant in the next 3        months, or lactating females.    -   b. History of genital herpes with >3 outbreaks per year, or        active non-HPV vaginal infection    -   c. Positive result for Hep B, Hep C or HIV.    -   d. Have an active pelvic infection (positive urine screen for        gonorrhoea or chlamydial infection, positive test and symptoms        for bacterial vaginosis, candida vaginitis or trichomonal        vaginitis)    -   e. Current or recent abnormal vaginal discharge and/or abnormal        vaginal bleeding, within the 3 months prior to randomization as        accessed by Investigator.    -   f. Had an abortion or miscarriage within the 3 months prior to        randomization    -   g. Currently taking any of the following medications: oral        corticosteroids, inhaled salmeterol and fluticasone;        immunomodulatory treatments, over the counter (OTC)        intra-vaginal preparation, or any prescription that in the        opinion of the Investigator could interfere with the        interpretation of the results.    -   h. Currently taking any of the medications listed        here—Alfuzosin, Amiodarone, dronedarone, Ranolazine, Fusidic        Acid, Colchicine, Astemizole, terfenadine, Lurasidone, Pimozide,        Quetiapine, Dihydroergotamine, ergonovine, ergotamine,        methylergonovine, Cisapride, Lovastatin, simvastatin, Avanafil,        Sildenafil, Vardenafil, Oral midazolam, triazolam, St. John's        wort.    -   i. Recent history (within previous 3 months) of Stevens-Johnson        syndrome, erythema multiforme, urticaria, angioedema, deep vein        thrombosis, tinnitus, vertigo, blood glucose disorders,        pancreatitis, haemophilia.    -   j. Hypersensitivity to any component of R131 vaginal ointment        excipients    -   k. Participation in any clinical study with an experimental        medication or device within 30 days or 5 half-lives (whichever        is longer) of enrolment.    -   l. Current alcohol or substance abuse as assessed by the        Investigator.    -   m. An employee or first-degree family member of an employee, the        Sponsor, the CRO or study site.    -   n. Not having a GP

Screening Evaluations:

The screening evaluations must have been made within 3 months ofrandomization into the study. Screening consisted of the followingcomponents:

Demographic/Medical History

A complete medical history was taken from each participant.

Physical Examination

The physical examination consisted of a review of body systems withheight and weight (in indoor clothing).

Blood Tests

The following laboratory blood tests are performed:

-   -   Electrolytes (sodium and potassium), ALT, GGT, ALP, albumin,        total protein, total bilirubin, urea, uric acid, serum        creatinine, TFT, fasting lipids, amylase, glucose, and HbA1c    -   Haemoglobin, red cell count, PCV, MCV, MCH, platelet count,        white cell count, neutrophils, lymphocytes, monocytes,        eosinophils and basophils. CD4/CD8 counts    -   HIV and hepatitis B and C.        The measurement at screening serve as a baseline to monitor any        abnormalities that may manifest as a result of dosing

Other Tests

Drugs of abuse testing were carried out on all participants as part ofthe screening procedures. A urine sample was required to test forcannabinoids (marijuana), amphetamines, benzodiazepines and opiates(i.e. morphine, heroin and codeine).Urinalysis dipstick to check for protein, leucocytes, nitrites, pH,specific gravity, glucose, ketones, and blood.Vaginal swabs for microbiology (gonorrhoea, Chlamydia, bacterialvaginosis, candida) and HPV genotypingAlcohol breath testing was carried out at the Clinical Site on the firstnight of each confinement period.Serum HCG testing was carried out on all participants as part of thescreening procedures and within 3 days before the 1st dose.

Vital Signs

Vital signs were recorded and consisted of blood pressure (supine andsitting), heart rate, temperature and respiratory rate. Participants'vital signs should be within the following limits:Heart rate ≥60 or ≤99 beats/minute

Supine: Systolic Blood Pressure ≥90 or ≤160 mm Hg; Diastolic BloodPressure ≥50 or ≤90 mm Hg Sitting: Systolic Blood Pressure ≥90 or ≤160mm Hg; Diastolic Blood Pressure ≥50 or ≤90 mm Hg Temperature ≥36° C. or≤37.7° C.

Respiratory Rate ≥12 or ≤20 breaths/minute

Summary of Study Activities/Schedule of Events

Informed consent was needed from each participant. Participants werescreened to confirm study eligibility.

Randomisation

Participants were randomized following the Principal Investigator ortheir delegates documented acceptance of participants following reviewof completed screening procedures.

Study Confinement

Participants arrived at approximately 5pm on Day 1 and Day 22. Theduration of study confinement was approximately 27 hours. Participantswere released from the clinical site once the 24-hour post doseassessments had been completed.

Dosing

Dosing began at approximately 8 μm on each day dosing was scheduled.Participants are instructed to insert the medication in private. Dosingapplicators were returned to study staff and examined to ensure the fulldose has been applied and for reconciliation of study drug.

Sample Collection

Vaginal Swabs were Self-Administered by the Participants.PK Blood samples: blood samples (8 mL) were drawn through venouscatheters and transferred into vacutainers containing sodium heparin asthe anti-coagulant. The time of collection is recorded as the time thefull 8 mL of blood was collected. The venous catheters were kept patentby flushing with 1.5 mL-2.0 mL of heparinized saline following eachsample (0-24 hours). The sampling intervals were at: Day 1-2: 0, 1, 2,4, 8, 12, 24 hours; Day 22-23: 0, 1, 2, 4, 8, 12, 24 hours. Samples werecollected at their due time. Any deviation should have been noted.

Sample Processing and Storage

Plasma: Plasma was separated by centrifugation at 3500 rpm for 5 minutesat about 4° C. No aids for separation of plasma from red cells was used.The plasma sample is transferred with clean pipettes. The assay wasdetermined using a validated Analytical method.Each plasma sample was placed into a polypropylene storage tube with ascrew cap. The plasma was stored frozen at −60° C. or colder at theclinical site pending transfer to a Laboratory for assay.

End of Study

Within one week after the last study day, each participant was requiredto provide a blood sample for analysis. Any abnormalities as compared toinitial screening were monitored and followed up until they return tonormal.Participants were assessed for the occurrence of adverse events fromconsent until the last study day in each cohort.Vital signs (blood pressure, heart rate, respiratory rate andtemperature) at last study visit.

-   -   Laboratory tests (haematology (CBC, CD4+/CD8+ peripheral        lymphocyte count, biochemistry (RFT, LFT, electrolytes, TFT,        fasting lipids, HbA1c, amylase), Serum HCG Pregnancy and        urinalysis (dipstick), at last study visit.    -   A follow-up phone call to each participant is made within 7 days        (+2 days) of the end of the study to record any possible Adverse        Events (AE) post study. Any events are recorded in source        documentation.        All AE's were followed-up until resolution, or until the        Investigator was of the opinion that follow-up was no longer        required, or until 30 days from the last dose (as long as the        Investigator was satisfied that follow-up is no longer        required), whichever is earlier.

Adverse Events

During confinement the designated Supervisor for the study or adelegated representative must have been present at the study sitethroughout the study. Principal Investigator or at least one delegatedTrial Physician was on call throughout the studies. On all study visitseach participant was asked how they felt. This occurred at each samplingpoint throughout the study. AE's were recorded in source documentation.Each AE was classified by the Principal Investigator as serious adverseevent (SAE) or non-serious. Non-serious adverse events were assessed asbeing mild, moderate, or severe to describe the maximum intensity of theAE. The Principal Investigator also provided the possible relationshipbetween the AE and the study medication as highly probable, probable,possible, remotely or not (“no”) related to the study medication.The Principal Investigator should have stated if the cause of the AE isrelated to the concurrent non-investigational medication(s) if any arebeing taken, an underlying disease, a combination of these factors or isunknown.

9.2 Results Safety Results:

-   -   109 adverse events in total reported by 18 participants, see        Table 7.    -   1 event ‘related’ to study medication    -   6 events ‘probably related’ to study medication    -   82 events ‘possibly related’ to study medication    -   6 events ‘probably not related’ to study medication    -   14 events ‘not related’ to study medication    -   No SAEs

Conclusion:

The formulations according to the invention are deemed to bewell-tolerated since all AEs were either minor or not related toadministration of study medication.

TABLE 7 Adverse event summary: Composition Composition Composition ofTable 2 of Table 6 of Table 3 AE (Cohort 1) (Placebo) (Cohort 2) Vaginaldischarge 5 4 5 Vaginal pruritis 4 5 3 Vulvovaginal 3 5 2 discomfortVaginal odour 1 2 2 Oligomenorrhoea 4 2 3 Pelvic pain 2 2 0 Vulvovaginal1 2 0 burning sensation Vulvovaginal pain 0 1 0 Dysuria 0 1 0 Abdominalpain 5 3 2 Nausea 0 1 0 Abdominal 0 0 1 distension Bacterial vaginosis 24 3 Vulvovaginal 4 4 1 candidiasis Rhinorrhoea 1 1 0 Nasopharyngitis 0 01 Headache 2 0 0 Insomnia 0 0 1 Catheter site pain 1 1 1 Dizziness 1 0 0Drug 1 0 0 hypersensitivity Decreased appetite 0 0 1

Pharmacokinetic Analyses:

Pharmacokinetic parameters:The area under the plasma drug concentration time curve (AUC), the peakplasma drug concentration (Cmax) and the time to maximum drugconcentration (Tmax) were determined for lopinavir and ritonavir foreach subject receiving active treatment.The plasma drug concentration (C) versus the real sampling time (t) datawere analysed by a “noncompartmental” method to obtain thepharmacokinetic parameters. Initially the plasma data in the postdistribution phase of the plasma concentration—time plot were fittedusing linear regression to:

In C=In Co−t.Kel

where Co is the zero-time intercept of the extrapolated terminal phaseand Kel is the terminal elimination rate constant.The area (AUC_(0-t)) from time zero to the last determinedconcentration-time point (t) in the post distribution phase wascalculated using the trapezoidal rule.

Lopinavir and Ritonavir Concentration and Pharmacokinetic Parameters

The mean lopinavir and ritonavir plasma concentration-time data for eachsampling time is listed in Tables 8 and 9. The pharmacokineticparameters for lopinavir and ritonavir are summarised in Tables 10 and11.

TABLE 8 Mean (±SD) Plasma Lopinavir and Ritonavir Concentration Data vsSampling Times (Composition of Table 2) Vaginal Ointment) LopinavirRitonavir Test Treatment B: Test Treatment B: Test Treatment B: TestTreatment B: Single Dose Multiple Dose Single Dose Multiple Dose 1 × 3 gof 150 mg 21 × 3 g of 150 mg 1 × 3 g of 150 mg 21 × 3 g of 150 mgComposition of Composition of Composition of Composition of Table 2Table 2 Table 2 Table 2 Vaginal Ointment Vaginal Ointment VaginalOintment Ointment (n = 6) (n = 5) (n = 6) (n = 5) Sample ConcentrationConcentration Concentration Concentration time Mean value Mean valueMean value Mean value (hrs) (pg/mL) S.D (pg/mL) S.D (pg/mL) (pg/mL) S.D0 0.0 0.0 115.1 64.3 0.0 0.0 3.7 8.2 1 28.9 35.9 115.8 50.1 2.7 6.5 0.00.0 2 49.6 44.0 169.6 76.4 3.0 7.4 3.9 8.6 4 94.3 79.7 194.1 77.5 35.866.4 12.5 11.7 8 103.7 60.3 170.4 43.4 9.4 14.6 9.9 9.0 12 132.1 78.2168.2 51.2 11.8 13.7 6.9 9.5 24 150.8 63.0 178.3 64.4 0.0 0.0 10.1 14.0

TABLE 9 Mean (±SD) Plasma Lopinavir and Ritonavir Concentration Data vsSampling Times (Composition of Table 3) Vaginal Ointment) LopinavirRitonavir Test Treatment A: Test Treatment A: Test Treatment A: TestTreatment A: Single Dose Multiple Dose Single Dose Multiple Dose 1 × 3 gof 300 mg 21 × 3 g of 300 mg 1 × 3 g of 300 mg 21 × 3 g of 300 mgComposition of Composition of Composition of Composition of Table 3Table 3 Table 3 Table 3 Vaginal Ointment Vaginal Ointment VaginalOintment Vaginal Ointment (n = 6) (n = 5) (n = 6) (n = 5) SampleConcentration Concentration Concentration Concentration time Mean valueMean value Mean value Mean value (hrs) (pg/mL) S.D (pg/mL) S.D (pg/mL)(pg/mL) S.D 0 0.0 0.0 334.7 310.3 0.0 0.0 40.2 43.1 1 55.5 73.1 288.9234.2 4.5 11.1 44.6 43.9 2 114.8 165.8 344.9 310.0 11.8 28.8 34.0 38.3 4181.4 241.0 323.2 252.9 12.3 30.1 44.0 31.9 8 198.7 181.8 307.4 211.614.7 20.5 37.3 21.8 12 179.4 141.8 337.7 218.4 16.0 22.0 39.5 24.3 24169.2 71.1 248.4 186.9 12.1 13.4 29.4 14.8

TABLE 10 Pharmacokinetic Parameters for Lopinavir Test Treatment B: TestTreatment B: Single Dose Multiple Dose 1 × 3 g of 150 mg 21 × 3 g of 150mg Composition of Table Composition of Table 2Vaginal Ointment 2VaginalOintment Pharmacokinetic (n = 6) (n = 5) Parameters (mean ± S.D) (Range)(mean ± S.D) (Range) AUC_(0-t) (pg · hr/ml) 1560.2 ± 989.7 2529.1 ±840.3 (491.1-3206.0) (1478.7-3614.2) Cmax (pg/ml) 181.8 ± 66.9 228.1 ±60.3 (72.0-269.2) (168.2-320.3) Tmax(hr) 18.72 ± 8.67  11.61 ± 11.36(4.00-24.23)  (2.00-24.03) Test Treatment A: Test Treatment A: SingleDose Single Dose 1 × 3 g of 300 mg 21 × 3 g of 300 mg Composition ofTable 3 Composition of Table 3 Vaginal Ointment Vaginal OintmentPharmacokinetic (n = 6) (n = 6) Parameters (mean ± S.D) (Range) (mean ±S.D) (Range) AUC_(0-t) (pg · hr/ml)  4021.1 ± 3093.5 7368.1 ± 4973.1(1710.9-9944.3)  (2366.4-13858.2) Cmax (pg/ml)  254.7 ± 211.7 396.3 ±297.3 (97.8-670.2) (130.2-840.7)  Tmax(hr) 16.03 ± 9.16 11.61 ± 8.06 (4.00-24.12) (2.00-24.03)

TABLE 11 Pharmacokinetic Parameters for Ritonavir Test Treatment B: TestTreatment B: Single Dose Multiple Dose 1 × 3 g of 150 mg 21 × 3 g of 150mg Composition of Table Composition of Table 2Vaginal Ointment 2VaginalOintment Pharmacokinetic (n = 6) (n = 5) Parameters (mean ± S.D) (Range)(mean± S.D) (Range) AUC_(0-t) (pg · hr/ml) 161.1 ± 254.9 165.0 ± 158.3(0.0-667.8) (16.5-407.8) Cmax (pg/ml) 45.5 ± 61.7 21.2 ± 5.1 (0.0-169.1) (15.6-27.9)  Tmax(hr) 5.33 ± 4.13 8.82 ± 8.71 (0.00-12.00) (4.00-24.08) Test Treatment A: Test Treatment A: Single Dose Single Dose1 x 3 g of 300 mg 21 x 3 g of 300 mg Composition of Table 3 Compositionof Table 3 Vaginal Ointment Vaginal Ointment Pharmacokinetic (n = 6) (n= 6 ) Parameters (mean ± S.D) (Range) (mean ± S.D) (Range) AUC_(0-t) (pg· hr/ml) 313.5 ± 447.6 890.0 ± 548.3  (0.0-1156.4) (420.6-1564.5) Cmax(pg/ml) 23.1 ± 27.2 53.3 ± 35.3 (0.0-73.8) (25.3-98.5)  Tmax(hr) 10.02 ±11.27 5.20 ± 4.77 (0.00-24.12) (1.00-12.02)

Discussion of Results: Cmax:

Following an oral dose of 400 mg lopinavir (as Kaletra 400 mg/100 mgtablets)* twice daily for 2 weeks, the mean Cmax for lopinavir was12.3±5.4 μg/mL (SPMC Kaletra). Adjusting for dose comparison with a 300mg dose administered topically in ointment form, the mean Cmax would be9.23±4.1 μg/mL.Following a topical dose of 300 mg lopinavir daily for 21 days as 2.5 gointment containing 12% w/w lopinavir, the mean Cmax was 396.3±297.3μg/mL.The ratio of Cmax oral/Cmax topical is >23,000 indicating that less than0.004% of the topical dose is available systemically.

AUC 0-t:

Following an oral dose of 400 mg lopinavir (as Kaletra 400 mg/100 mgtablets)* twice daily for 2 weeks, the AUC0-t for lopinavir was113.2±60.5 μg·h/mL (SPMC Kaletra). Adjusting for dose comparison with a300 mg dose administered topically in ointment form, the AUC0-t would be84.9±45.4 μg·h/mL.Following a topical dose of 300 mg lopinavir daily for 21 days as 2.5 gointment containing 12% w/w lopinavir, the AUC0-t was 7368.1±4973.1pg/mL.The ratio of AUC oral/AUC topical is >11,500 indicating that less than0.009% of the topical dose is available systemically.

Conclusion:

The combined Cmax and AUC data indicate that systemic absorption oflopinavir from topical administration of the ointment is negligible.

Example 10 A Phase 1b, Multicentre, Open Label, Study of the Efficacy,Safety and Tolerability of a Composition comprising Ritonavir andLopinavir in Women with Cytological Abnormalities of the Uterine Cervix

10.1 Composition usedThe composition according to Table 5 was used in a Phase 1b trialexcept, rather than employing the methodology of Example 7, a coldprocess was employed as described below.The manufacture of a vaginal dosage form exhibiting rheology suitablefor syringe applicator vaginal dosing is described below in accordancewith Table 5:

-   -   i. Into medicine mixer add 1, 9, 10, 11    -   ii. Lower the lid and purge with nitrogen    -   iii. Mix without heating for several hours until a clear,        transparent solution is achieved    -   iv. Raise the lid and add 7    -   v. Lower the lid and purge with nitrogen    -   vi. Mix without heating until a clear, transparent solution is        achieved    -   vii. Raise the lid and add 2, 3, 4, 5, 6, 8    -   viii. Lower the lid and purge with nitrogen    -   ix. Mix without heating for several hours    -   x. Discharge the product into bulk storage vessel, awaiting        packing into aluminium tubes

10.2 Methodology Study Objectives Efficacy Objectives

-   -   Demonstrate histological clearance of cytological abnormalities        following application of a composition according to the        invention in women with high-grade or low-grade CIN (Cervical        intra-epithelial neoplasia).    -   Demonstrate changes to colposcopic appearance of the uterine        cervix following application of a composition according to the        invention;    -   Assess changes in HPV status following application of a        composition according to the invention.

Safety Objective

-   -   Assess the incidence of AEs following the application of a        composition according to the invention.

Tolerability Objective

-   -   Assess the tolerability of a composition according to the        invention, measured by compliance with dosing schedule of a        composition according to the invention during 21 consecutive        days of treatment for up to 3 treatment cycles.

Study Design:

This study is designed as a Phase 1b multicentre, open label studyinvestigating the efficacy, safety and tolerability of a compositionaccording to the invention in women with cytological abnormalities ofthe uterine cervix.In this single arm study, participants are stratified according to theirgrade of cytological abnormality:

-   -   Biopsy proven high-grade cytological abnormalities of the        uterine cervix defined as CIN 2 and above;    -   Low-grade cytological abnormalities of the uterine cervix        defined as CIN 1/LSIL.        The composition according to the invention is self-applied to        the vagina once a day for 21 consecutive days in up to 3        treatment cycles. Participants attend a clinic visit 7 days        after administration of the final dose of investigational        product. Participants complete a daily diary card and Vaginal        Irritation Questionnaire to capture compliance with        investigational product administration, AEs and changes to        concomitant medication.        Participation in this study includes a screening visit, up to 3        treatment cycles and an end of study visit as follows:    -   Screening Visit: Day −28 to Day 0.    -   Treatment Cycle 1:        Day 1* to Day 21: Treatment Cycle 1—investigational product        applied once daily for 21 days;        Day 8, 15, 22: telephone follow up; check AEs, conmeds,        compliance, dosing issues;        Day 28: visual assessment of disease, if no disease detected#,        treatment is stopped, and a biopsy is performed 6 weeks later        (Day 70).    -   Treatment Cycle 2:        Day 29** to Day 49: Treatment Cycle 2—non-responders, identified        at Day 28, will continue investigational application once daily        for 21 days;        Day 36, 43, 50: telephone follow up; check AEs, conmeds,        compliance, dosing issues;        Day 56: visual assessment of disease, if no disease detected#,        treatment is stopped, and a biopsy is performed 6 weeks later        (Day 98).    -   Treatment Cycle 3:        Day 57** to Day 77: Treatment Cycle 3 non-responders, identified        at Day 56, can continue investigational application once daily        for 21 days OR be referred to their primary physician;        Day 64, 71, 78: telephone follow up; check AEs, conmeds,        compliance, dosing issues;        Day 84: visual assessment of disease and biopsy performed 6        weeks later (Day 126)    -   Post-treatment assessment visit (PTAV)/Early termination visit        (ETV): Day 70, Day 98 or Day 126 depending on response.        *Day 1 commences at the end of a participant's menstrual cycle.        ** Day 29 and 57 may be delayed to the end of the participants'        menstrual cycle if required.        #If absence of disease, defined as no colposcopic evidence of        CIN, participants are considered responders. If disease is        detected, defined as ongoing colposcopic evidence of CIN,        participants are considered non-responders.

Participation Criteria Inclusion Criteria:

To be eligible for study entry participants must satisfy all of thefollowing criteria:

-   1. Provision of written informed consent prior to any study specific    procedures;-   2. Female participants aged 25-45 years inclusive at the time of    screening visit;-   3. Positive result for cervical high-risk HPV (types 16, 18 or    ‘other’);-   4. High-grade cytological abnormality of the uterine cervix defined    as CIN 2 as proven by colposcopic biopsy collected at screening

OR

-   -   low-grade cytological abnormality of the uterine cervix defined        as CIN 1/LSIL, as demonstrated by colposcopic biopsy within 6        months prior to screening.        Participants will be stratified according to their grade of        cytological abnormality;

-   5. Transformation zone needs to be fully visible;

-   6. Generally, in good health with no clinically significant disease    as determined by the investigator;

-   7. Regular menstrual cycle with an approximate 28-day cycle

OR

-   -   women who are amenorrhoeic due to effective contraception (such        as Mirena, Jadelle, or continuous COC)

-   8. Agree to abstain from activities such as vaginal douching or    insertion of any vaginal products other than the study drug for at    least 48 hours prior to enrolment and throughout the study. Tampons    may be used during the menstrual cycle only.

-   9. Women of childbearing potential (WOCBP) must use a highly    effective form of birth control (confirmed by the Investigator).    Rhythm methods will not be considered as highly effective methods of    birth control. Highly effective forms of birth control include:    -   True sexual abstinence (defined as refraining from heterosexual        intercourse for the duration of the study and a minimum of 30        days following the last dose of study drug);    -   Vasectomised partner (provided that the partner is the sole        sexual partner of the female participant with childbearing        potential and that the vasectomised partner has received medical        assessment of the surgical success);    -   Oral or transdermal combined (oestrogen and progestogen        containing) hormonal contraception associated with inhibition of        ovulation;    -   Oral, injectable or implantable progestogen-only hormone        contraception associated with inhibition of ovulation        (Depo-Provera™, Implanon);    -   Any effective intrauterine device/levonorgestrel intrauterine        system;    -   Female sterilisation by tubal occlusion;    -   Evra Patch™.        WOCBP must agree to use a highly effective method of birth        control, as defined above, from enrolment, and at least 14 days        prior to Day 1, throughout the study duration and within 30 days        after the last dose of IMP.        WOCBP are defined as women who are neither permanently        sterilised (hysterectomy, bilateral oophorectomy, or bilateral        salpingectomy), nor who are postmenopausal. Women will be        considered post-menopausal if they have been amenorrhoeic for 12        months or more without an alternative biological or medical        cause e.g. contraceptive method such as Mirena.

-   10. Male partners of female participants must agree to use condoms    during sexual intercourse from the first dose of investigational    product until 30 days after the participants last dose to avoid    potential transfer of investigational product.

-   11. Able and willing to abstain from sexual intercourse from 6 hours    prior to dosing until 6 hours after dosing;

-   12. Ability and willingness to attend the necessary visits to the    study centre;

-   13. Ability to comprehend all study related documentation, including    written informed consent form, and complete all study-related tasks    including daily diary;

-   14. Be willing and able to adhere to the prohibitions and    restrictions specified in the protocol.

Exclusion Criteria:

Participants are excluded from the study if one or more of the followingcriteria are applicable:

-   1. Any significant disease or disorder (e.g. cardiovascular,    pulmonary, gastrointestinal, hepatic, renal, neurological,    musculoskeletal, endocrine, metabolic, malignant, psychiatric, major    physical impairment) which, in the opinion of the investigator, may    either put the participant at risk because of participation in the    study, or may influence the results of the study, or the    participant's ability to participate in the study;-   2. Any clinically significant abnormal findings in physical    examination, vital signs, haematology, clinical chemistry, or    urinalysis during screening and at baseline, which in the opinion of    the investigator, may put the participant at risk because of her    participation in the study, or may influence the results of the    study, or the participant's ability to complete entire duration of    the study;-   3. Pregnant, breastfeeding, or lactating women (WOCBP must have a    negative serum pregnancy test at screening and a negative urine    pregnancy test at the start of each treatment period [i.e. Day 1,    Day 28, Day 56]);-   4. Women who plan to become pregnant in the next 6 months;-   5. History of genital herpes with >3 outbreaks per year, or active    non-HPV vaginal infection;-   6. Active pelvic infection (positive for gonorrhoea or chlamydial    infection, positive test for bacterial vaginosis, candida vaginitis    or trichomonal vaginitis). Participants with positive results can be    re tested once during screening;-   7. Positive bimanual exam consistent with pelvic inflammatory    disease;-   8. Positive result for hepatitis B, hepatitis C or human    immunodeficiency virus;-   9. Current or recent abnormal vaginal discharge and/or abnormal    vaginal bleeding, within the 3 months prior to Day 1 as assessed by    the investigator;-   10. Had an abortion or miscarriage or taken the morning-after pill    within the 3 months prior to enrolment;-   11. Currently taking immunosuppressants, intra-vaginal preparations,    or any prescription that in the opinion of the investigator could be    a potential safety issue or interfere with the interpretation of the    results;-   12. Previous exposure to lopinavir/ritonavir (within 3 months prior    to screening), contraindication to the use of lopinavir/ritonavir or    known allergy, hypersensitivity, or intolerance to any component of    lopinavir/ritonavir ointment excipients;-   13. Previous HPV vaccination;-   14. Recent history (within 3 months prior to screening) of    Stevens-Johnson syndrome, erythema multiforme, urticaria,    angioedema, deep vein thrombosis, tinnitus, vertigo, blood glucose    disorders, pancreatitis, haemophilia;-   15. Receipt of any investigational product within 30 days or 5    half-lives prior to dosing;-   16. Employees of the clinical study team or family members    (first-degree relatives) of such individuals or anyone involved in    the planning and/or conduct of the study. Clinical study team refers    to employees directly involved in the study who have been delegated    study-related tasks accordingly;-   17. Participants who, in the opinion of the Investigator, do not    understand the information and procedures of the study, or would not    be compliant with them (in particular the study restrictions and    risks involved).

Dosing Schedule:

Investigational product is administered every day for 21 days for up to3 cycles as follows:

-   -   Cycle 1 (daily at Bpm from Day 1 to Day 21);    -   Cycle 2 (daily at Bpm from Day 29 to Day 49);    -   Cycle 3 (daily at Bpm from Day 57 to Day 77).        There is a 7-day treatment cessation in between cycles to allow        for menstruation.        Investigational product is self-administered by participants at        approximately 8pm (±1 hour) each evening. Participants are        provided dosing instructions and important application        instructions.        Participants record details of investigational product        application in a diary card each day to monitor compliance.        Participants are also asked to note in their diary card if there        is any waste/spillage.        Participants are required to bring their investigational product        and diary card to each study site visit. The tubes are to be        weighed prior to dispensing to the participant and again at each        clinic visit to assess compliance.        There are no fasting requirements associated with the timing of        application of investigational product.

Safety Assessments

The following safety assessments are performed at timepoints outlined inthe Schedule of Events, see Table 12.

-   -   Medical History    -   Physical Examination    -   Vital Signs    -   Body Weight and Height    -   12-lead Electrocardiogram    -   Clinical Laboratory Safety Tests (Haematology, Biochemistry,        Urinalysis, Vaginal Microbiology, Viral Serology, Drugs of Abuse        Screen, Alcohol Screen, Pregnancy Screen)

Efficacy Assessments

The efficacy of the investigational product are assessed by improvementsin the cytological abnormalities of the uterine cervix. Efficacyassessments are performed at timepoints outlined in the Schedule ofEvents, see Table 12.

-   -   Colposcopic visual assessment    -   Colposcopic Biopsy    -   Cytological Sampling    -   HPV Genotyping

TABLE 12 Treatment Cycle 2 Treatment Cycle 3 Post-treatment TreatmentCycle 1 (Treatment Cycle 1 (Treatment Cycle 1 and assessment/EarlyScreening (all participants) non-responders only) 2 non-responders only)Termination Visit Day 6 weeks after −28 to last dose of 0 1 8 15 22 2829 36 43 50 56 57 64 71 78 84 Composition Attend study site X X X X X XTelephone X X X X X X X X X Follow up Informed consent X Inclusion/ X Xexclusion criteria Demographics X Medication X history Physical X Xexamination Vital signs X X X X X X Weight, height X 12-lead Safety XECG Alcohol breath X test Drugs of abuse X X screen Concomitant X X X XX X X X X X X X X X X X X medications Adverse events X X X X X X X X X XX X X X X X X Haematology X X X X X Biochemistry X X X X X Serology XUrinalysis X X X X X Pregnancy Test X X X X X Vaginal X X X X Xmicrobiology Vaginal pH X X X X X Papanicolaou X X smear HPV genotypingX X Colposcopy X X X X X visual assessment Colposcopic X X biopsyInvestigational X X X product dispensed compound X X X X X X X X Xadministration Participant diary/ X- — — — X — — — — X — — — — X VIQcompletion — Review X X X participant diary card and questionnaireReview X X X investigational product

10.3 Results

Preliminary results for a subject with CIN1 at screening (who received21 days of composition, no missed doses):

-   -   (A) Subject had slight irritation and vaginal itchiness thought        out the 21 days but this subsided.    -   (B) Subject had colposcopy visual assessment after 21 days and        no lesions could be detected.        Plan: as the condition had regressed the clinical assess felt        there was no need to continue to cycle 2. Subject will be        followed with end of study appointment

1. A pharmaceutical composition that is formulated for topicalapplication comprising a therapeutically effective amount of lopinavirand ritonavir in a pharmaceutically acceptable vehicle and wherein theweight ratio (w/w) of lopinavir: ritonavir is between 9:1 and 18:1. 2.The composition according to claim 1 for use as a medicament fortreating and/or inhibiting the development or progression of cervicalcancers and benign proliferative disorders of the cervix.
 3. (canceled)4. (canceled)
 5. (canceled)
 6. The composition according to claim 1wherein lopinavir and ritonavir are included in a weight ratio (w/w) ofbetween 10:1 and 16:1.
 7. The composition according to claim 6 whereinlopinavir and ritonavir are included in a weight ratio of about11:1-16:1.
 8. The composition according to claim 7 wherein lopinavir andritonavir are included in a weight ratio of about 12:1.
 9. Thecomposition according to claim 1 wherein the composition is formulatedas an ointment, gel, paste, cream, lotion, ovule, soft capsule,suppository, pessary, or any combination thereof.
 10. The compositionaccording to claim 1 wherein the pharmaceutically acceptable vehicle isan ointment.
 11. The composition according to claim 10 wherein thecomposition is an anhydrous composition for topical applicationcomprising: (a) Lopinavir and ritonavir in a weight ratio of between 9:1and 18:1; and (b) a hydrophilic muco-adhesive agent; wherein upontopical administration of the anhydrous composition to a site ofapplication the anhydrous composition transforms into a muco-adhesivecomposition.
 12. The composition according to claim 11 wherein themuco-adhesive agent is hydroxypropylmethylcellulose.
 13. The compositionaccording to claim 11 comprising ritonavir, lopinavir,hydroxypropylmethylcellulose, oleic acid, stearic acid; and butylatedhydroxytoluene.
 14. The composition according to claim 10 wherein thecomposition comprises: (a) an unsaturated free fatty acid; (b) astiffening agent; and (c) lopinavir and ritonavir in a weight ratio ofbetween 9:1 and 18:1; wherein the unsaturated free fatty acid is presentat a level of at least 20% by weight of the total pharmaceuticalcomposition weight and wherein the pharmaceutical composition is asemi-solid at room temperature.
 15. A method of treating and/orinhibiting the development or progression of cervical cancers and benignproliferative disorders of the cervix in a subject in need of suchtreatment or inhibition comprising administering a therapeuticallyeffective amount of a pharmaceutical composition according to claim 1 tosaid subject.
 16. The method according to claim 15 wherein the cancer ordisorder is caused or induced by a human papilloma virus (HPV).
 17. Amethod of treating a patient having an HPV related dysplasia of thecervix comprising administering intravaginally to said patient atherapeutically effective dose of a pharmaceutical composition accordingto claim
 1. 18. The method of claim 17, wherein the pharmaceuticalcomposition reduces the severity of the HPV related dysplasia.
 19. Themethod of claim 18, wherein the severity of the HPV related dysplasia isreduced from CIN3 to CIN2, from CIN3 to CIN1, from CIN3 to HPV negative,from CIN2 to CIN1, from CIN2 to HPV negative, or from CIN1 to HPVnegative.
 20. The method of claim 16, wherein the composition inducesapoptosis of HPV infected cells.
 21. The method of claim 15, wherein thepatient has a cervical cytology of high grade squamous intraepitheliallesion (HSIL), atypical squamous cells of undetermined significance(ASCUS), or low grade squamous intraepithelial lesion (LSIL).
 22. Themethod of claim 21, wherein the composition reduces the cervicalcytology from HSIL to a normal cytology, from HSIL to ACSUS, from HSILto LSIL, from ACSUS to a normal cytology, or from LSIL to a normalcytology.
 23. A method of treating a Human Papilloma Virus (HPV)infection with or without attendant abnormal pathology, comprisingadministering an effective amount of the pharmaceutical composition ofclaim 1.